Methods for treating obesity using fibroblast growth factor-Like polypeptides

ABSTRACT

The present invention provides novel Fibroblast Growth Factor-like (FGF-like) polypeptides and nucleic acid molecules encoding the same. The invention also provides vectors, host cells, antibodies and methods for producing FGF-like polypeptides. Also provided for are methods for the diagnosis and treatment of diseases associated with FGF-like polypeptides.

FIELD OF THE INVENTION

The present invention relates to novel Fibroblast Growth Factor-like(FGF-like) polypeptides and nucleic acid molecules encoding the same.The invention also relates to vectors, host cells, antibodies andmethods for producing FGF-like polypeptides. Also provided for aremethods for the diagnosis and treatment of diseases associated withFGF-like polypeptides.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression, andmanipulation of nucleic acid molecules have greatly accelerated thediscovery of novel therapeutics based upon deciphering the human genome.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates and, coupled with computationalanalyses, allow the assembly of overlapping sequences into entiregenomes and the identification of polypeptide-encoding regions.Comparison of a predicted amino acid sequence against a databasecompilation of known amino acid sequences can allow one to determine theextent of homology to previously identified sequences and/or structurallandmarks. Cloning and expression of a polypeptide-encoding region of anucleic acid molecule provides a polypeptide product for structural andfunctional analysis. Manipulation of nucleic acid molecule and encodedpolypeptides to give variants and derivatives thereof may conferadvantageous properties on a product for use as a therapeutic.

In spite of the significant technical advances in genome research overthe past decade, the potential for development of novel therapeuticsbased on the human genome is still largely unrealized. Genes encodingpotentially beneficial protein therapeutics, or those encodingpolypeptides that may act as “targets” for therapeutic molecules, havestill not been identified. In addition, structural and functionalanalyses of polypeptide products from many human genes have not beenundertaken.

Accordingly, it is an object of the invention to identify novelpolypeptides and nucleic acid molecules encoding the same which havediagnostic or therapeutic benefit.

SUMMARY OF THE INVENTION

The present invention relates to novel FGF-like nucleic acid moleculesand encoded polypeptides.

The invention provides for an isolated nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:3;

(b) the nucleotide sequence of the DNA insert in ATCC Deposit No.PTA-626;

(c) a nucleotide sequence encoding the polypeptide as set forth in SEQID NO: 2 or SEQ ID NO: 4;

(d) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(c); and

(e) a nucleotide sequence complementary to any of (a)-(c).

The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide that is at least about80 percent identical to the polypeptide as set forth in SEQ ID NO: 2 orSEQ ID NO: 4, wherein the encoded polypeptide activates one or more FGFreceptors, regulates the growth and differentiation of cells within theliver or pancreas, regulates other cell types following secretion fromthe liver or pancreas, plays a role in liver or pancreas chemotaxis, orhas an oncogenic activity;

(b) a nucleotide sequence encoding an allelic variant or splice variantof the nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3,or (a);

(c) a region of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,(a), or (b) encoding a polypeptide fragment of at least about 25 aminoacid residues, wherein the encoded polypeptide activates one or more FGFreceptors, regulates the growth and differentiation of cells within theliver or pancreas, regulates other cell types following secretion fromthe liver or pancreas, plays a role in liver or pancreas chemotaxis, hasan oncogenic activity, or serves as an antigen for generatingantibodies;

(d) a region of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,or any of (a)-(c) comprising a fragment of at least about 16nucleotides;

(e) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(d); and

(f) a nucleotide sequence complementary to any of (a)-(d).

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 2 with at least one conservative amino acid substitution, whereinthe encoded polypeptide activates one or more FGF receptors, regulatesthe growth and differentiation of cells within the liver or pancreas,regulates other cell types following secretion from the liver orpancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity;

(b) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 2 with at least one amino acid insertion, wherein the encodedpolypeptide activates one or more FGF receptors, regulates the growthand differentiation of cells within the liver or pancreas, regulatesother cell types following secretion from the liver or pancreas, plays arole in liver or pancreas chemotaxis, or has an oncogenic activity;

(c) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 2 with at least one amino acid deletion, wherein the encodedpolypeptide activates one or more FGF receptors, regulates the growthand differentiation of cells within the liver or pancreas, regulatesother cell types following secretion from the liver or pancreas, plays arole in liver or pancreas chemotaxis, or has an oncogenic activity;

(d) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 2 which has a carboxyl- and/or amino-terminal truncation, whereinthe encoded polypeptide activates one or more FGF receptors, regulatesthe growth and differentiation of cells within the liver or pancreas,regulates other cell types following secretion from the liver orpancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity;

(e) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 2 with at least one modification selected from the group consistingof amino acid substitutions, amino acid insertions, amino aciddeletions, carboxyl-terminal truncation, and amino-terminal truncation,wherein the encoded polypeptide activates one or more FGF receptors,regulates the growth and differentiation of cells within the liver orpancreas, regulates other cell types following secretion from the liveror pancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity;

(f) a region of the nucleotide sequence of any of (a)-(e) comprising afragment of at least about 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(f); and

(h) a nucleotide sequence complementary to any of (a)-(e).

The present invention provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO:4; and

(b) the amino acid sequence encoded by the DNA insert of ATCC DepositNo. PTA-626.

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in either SEQ ID NO: 5 or SEQID NO: 6, optionally further comprising an amino-terminal methionine;

(b) an amino acid sequence for an ortholog of either SEQ ID NO: 2 or SEQID NO: 4, wherein the encoded polypeptide activates one or more FGFreceptors, regulates the growth and differentiation of cells within theliver or pancreas, regulates other cell types following secretion fromthe liver or pancreas, plays a role in liver or pancreas chemotaxis, orhas an oncogenic activity;

(c) an amino acid sequence that is at least about 80 percent identicalto the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 4,wherein the encoded polypeptide activates one or more FGF receptors,regulates the growth and differentiation of cells within the liver orpancreas, regulates other cell types following secretion from the liveror pancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity;

(d) a fragment of the amino acid sequence set forth in either SEQ ID NO:2 or SEQ ID NO: 4 comprising at least about 25 amino acid residues,wherein the encoded polypeptide activates one or more FGF receptors,regulates the growth and differentiation of cells within the liver orpancreas, regulates other cell types following secretion from the liveror pancreas, plays a role in liver or pancreas chemotaxis, has anoncogenic activity, or serves as an antigen for generating antibodies;and

(e) an amino acid sequence for an allelic variant or splice variant ofeither the amino acid sequence as set forth in either SEQ ID NO: 2 orSEQ ID NO: 4; the amino acid sequence encoded by the DNA insert of ATCCDeposit No. PTA-626; (a), (b), or (c).

The invention further provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 4 with at least one conservative amino acid substitution, whereinthe encoded polypeptide activates one or more FGF receptors, regulatesthe growth and differentiation of cells within the liver or pancreas,regulates other cell types following secretion from the liver orpancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity;

(b) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 4 with at least one amino acid insertion, wherein the encodedpolypeptide activates one or more FGF receptors, regulates the growthand differentiation of cells within the liver or pancreas, regulatesother cell types following secretion from the liver or pancreas, plays arole in liver or pancreas chemotaxis, or has an oncogenic activity;

(c) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 4 with at least one amino acid deletion, wherein the encodedpolypeptide activates one or more FGF receptors, regulates the growthand differentiation of cells within the liver or pancreas, regulatesother cell types following secretion from the liver or pancreas, plays arole in liver or pancreas chemotaxis, or has an oncogenic activity;

(d) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 4 which has a C- and/or N-terminal truncation, wherein theencoded polypeptide activates one or more FGF receptors, regulates thegrowth and differentiation of cells within the liver or pancreas,regulates other cell types following secretion from the liver orpancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity; and

(e) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQID NO: 4 with at least one modification selected from the groupconsisting of amino acid substitutions, amino acid insertions, aminoacid deletions, C-terminal truncation, and N-terminal truncation,wherein the encoded polypeptide activates one or more FGF receptors,regulates the growth and differentiation of cells within the liver orpancreas, regulates other cell types following secretion from the liveror pancreas, plays a role in liver or pancreas chemotaxis, or has anoncogenic activity.

The invention also provides for an expression vector comprising thenucleic acid molecules as set forth above, host cells comprising theexpression vectors of the invention, and a method of production of anFGF-like polypeptide comprising culturing the host cells and optionallyisolating the polypeptide so produced.

A transgenic non-human animal comprising a nucleic acid moleculeencoding an FGF-like polypeptide is also encompassed by the invention.The FGF-like nucleic acid molecules are introduced into the animal in amanner that allows expression and increased levels of an FGF-likepolypeptide, which may include increased circulating levels.Alternatively, the FGF-like nucleic acid molecules are introduced intothe animal in a manner that prevents expression of endogenous FGF-likepolypeptide (i.e., generates a transgenic animal possessing an FGF-likepolypeptide gene knockout). The transgenic non-human animal ispreferably a mammal, and more preferably a rodent, such as a rat or amouse. Preferably the FGF-like transgene is expressed in the liver underthe control of the apolioprotein E promoter, or ubiquitously under thecontrol of the beta actin promoter.

Also provided are derivatives of the FGF-like polypeptides of theinvention, fusion polypeptides comprising the FGF-like polypeptides ofthe invention, and antibodies specifically binding the FGF-likepolypeptides of the invention.

Compositions comprising the nucleotides or polypeptides of the inventionand a carrier, adjuvant, solubilizer, stabilizer or anti-oxidant, orother pharmaceutically acceptable agent are also encompassed by theinvention. The compositions may include pharmaceutical compositionscomprising therapeutically effective amounts of the nucleotides orpolypeptides of the invention, and methods of using the polypeptides andnucleic acid molecules.

Surprisingly, FGF-like polypeptide appeared to be primarily expressed inthe liver (Northern analysis) and pancreatic islets (in situ analysis),thereby distinguishing it from all other members of the FGF family. Thepresent polypeptide, and its useful nucleic acid intermediates, may haveutility, therefore, in differentiating liver cells or pancreatic isletcells from background. Further, given the localization of FGF-likepolypeptide expression, the structural similarity of FGF-likepolypeptide to members of the FGF family, and the likelihood thatFGF-like polypeptide is secreted into the bloodstream where it may exerteffects on distal sites, the present polypeptides may provide benefitsin the stimulation of cells within or near the liver, regulation ofintestinal cell activity, stimulation of cells within or near pancreaticislets, regulation of neuronal cells, stimulation or inhibition ofangiogenesis, stimulation of epithelium or mesenchymal components ofgranulation tissue, stimulation of corneal epithelium, lens, or retinaltissue, regeneration of renal tubules, hematopoietic cell regulation,regulation of hair follicle growth, regulation of pulmonary epithelium,or stimulation of either epithelial, mesenchymal, hematopoietic, orneuronal cells or tissues, particularly as a therapeutic pharmaceuticalcomposition.

FGF-like polypeptides may also be useful as growth or fat depositioninhibitors, and therefore may be useful in the treatment of excessivegrowth (for example, acromegaly), premature maturation, obesity ordiabetes. Inhibitors—such as antibodies, binding proteins or smallmolecules—that interfere with the interaction of FGF-like polypeptidesand their receptor(s) may be useful in stimulating body growth andmaturation. Therefore, such inhibitors may be useful in treating shortstature, delayed maturation, or other conditions generally associatedwith impairment of signaling of growth hormone or its mediator,insulin-like growth factor.

The FGF-like polypeptides and nucleic acid molecules of the invention,or agonists or antagonists of their biological activity, may be used fortherapeutic or diagnostic purposes to treat, prevent and/or detect amedical condition such as cirrhosis or other toxic insult of the liver;inflammatory bowel disease, mucositis, Crohn's disease, or othergastrointestinal abnormality; diabetes; obesity; neurodegenerativediseases; wounds; damage to the corneal epithelium, lens, or retinaltissue; damage to renal tubules as a result of acute tubular necrosis;hematopoietic cell reconstitution following chemotherapy; wastingsyndromes (for example, cancer associated cachexia), multiple sclerosis,myopathies; short stature, delayed maturation, excessive growth (forexample, acromegaly), premature maturation; alopecia; diseases orabnormalities of androgen target organs; infantile respiratory distresssyndrome, bronchopulmonary dysplasia, acute respiratory distresssyndrome, or other lung abnormalities; tumors of the eye or othertissues; atherosclerosis; hypercholesterolemia; diabetes; obesity;stroke; osteoporosis; osteoarthritis; degenerative joint disease; muscleatrophy; sarcopenia; decreased lean body mass; baldness; wrinkles;increased fatigue; decreased stamina; decreased cardiac function; immunesystem dysfunction; cancer; Parkinson's disease; senile dementia;Alzheimer's disease; and decreased cognitive function. The inventionprovides for treating, preventing or ameliorating a disorder comprisingadministering to an animal an FGF-like polypeptide. The invention alsoprovides for a method of diagnosing such a disorder or a susceptibilityto such a disorder in an animal which includes both determining thepresence or amount of expression of an FGF-like polypeptide anddiagnosing such a disorder or a susceptibility to such a disorder basedon the presence or amount of expression of an FGF-like polypeptide. Theanimal is preferably a mammal, and more preferably a human. The presentinvention also relates to methods for the manufacture of a medicamentfor the treatment of a disorder such as those mentioned above.

The invention also provides for the use of antibodies or otherinhibitors of the binding of FGF-like polypeptide to its receptor forthe treatment of the same diseases listed above, and for the treatmentof tumors.

The invention also provides for a method of identifying a test moleculewhich binds to an FGF-like polypeptide wherein the method comprisescontacting an FGF-like polypeptide with a test molecule and determiningthe extent of binding of the test molecule to the polypeptide. Themethod further comprises determining whether such test molecules areagonists or antagonists of an FGF-like polypeptide.

The invention also provides for a method of testing the impact ofmolecules on the expression of FGF-like polypeptide or on the activityof FGF-like polypeptide.

A method of regulating expression and modulating (i.e., increasing ordecreasing) levels of an FGF-like polypeptide are also encompassed bythe invention. One method comprises administering to an animal a nucleicacid molecule encoding an FGF-like polypeptide. In another method, anucleic acid molecule comprising elements that regulate expression of anFGF-like polypeptide may be administered. Examples of these methodsinclude gene therapy and anti-sense therapy.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the nucleotide sequence of the murine FGF-like gene(SEQ ID NO: 3) and the deduced amino acid sequence of murine FGF-likeprotein (SEQ ID NO: 4);

FIGS. 2A-2B illustrate the nucleotide sequence of the human FGF-likegene (SEQ ID NO: 1) and the deduced amino acid sequence of humanFGF-like protein (SEQ ID NO: 2);

FIGS. 3A-3D illustrate the amino acid sequence alignment of humanFGF-like protein (hAgp-26257; SEQ ID NO: 2), murine FGF-like protein(mAgp-26257; SEQ ID NO: 4), human FGF-14 (Hfgf14; SEQ ID NO: 16), murineFGF-14 (Mfgf14; SEQ ID NO: 26), human FGF-12 (Hfgf12; SEQ ID NO: 15),murine FGF-13 (Mfgf13; SEQ ID NO: 25), human FGF-5 (Hfgf5; SEQ ID NO:20), murine FGF-5 (Mfgf5; SEQ ID NO: 30), human FGF-6 (Hfgf6; SEQ ID NO:21), murine FGF-6 (Mfgf6; SEQ ID NO: 31), human FGF-4 (Hfgf4; SEQ ID NO:19), murine FGF-4 (Mfgf4; SEQ ID NO: 29), human FGF-3 (Hfgf3; SEQ ID NO:18), murine FGF-3 (Mfgf3; SEQ ID NO: 28), human FGF-7 (Hfgf7; SEQ ID NO:22), murine FGF-7 (Mfgf7; SEQ ID NO: 32), human FGF-9 (Hfgf9; SEQ ID NO:23), murine FGF-9 (Mfgf9; SEQ ID NO: 33), human FGF-1 (Hfgf1; SEQ ID NO:14), murine FGF-1 (Mfgf1; SEQ ID NO: 24), human FGF-2 (Hfgf2; SEQ ID NO:17), murine FGF-2 (Mfgf2; SEQ ID NO: 27), and the resulting FGFconsensus sequence (cons);

FIGS. 4A-4C illustrate the results of (A) a Northern blot analysis ofmurine FGF-like polypeptide expression, (B) a Northern blot analysis ofhuman FGF-like polypeptide expression, and (C) a dot blot analysis ofhuman FGF-like polypeptide expression.

DETAILED DESCRIPTION OF THE INVENTION

The section headings herein are for organizational purposes only and arenot to be construed as limiting the subject matter described therein.All references cited in this application are expressly incorporated byreference herein.

DEFINITIONS

The term “FGF-like nucleic acid molecule” refers to a nucleic acidmolecule comprising or consisting essentially of a nucleotide sequenceas set forth in SEQ ID NO: 1 or SEQ ID NO: 3, comprising or consistingessentially of a nucleotide sequence encoding the polypeptide as setforth in SEQ ID NO: 2 or SEQ ID NO: 4, comprising or consistingessentially of a nucleotide sequence of the DNA insert in ATCC DepositNo. PTA-626, or nucleic acid molecules related thereto.

Related nucleic acid molecules comprise or consist essentially of anucleotide sequence that is about 80 percent identical to the nucleotidesequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3, or comprise orconsist essentially of a nucleotide sequence encoding a polypeptide thatis about 80 percent identical to the polypeptide as set forth in SEQ IDNO: 2 or SEQ ID NO: 4. In preferred embodiments, the nucleotidesequences are about 85 percent, or about 90 percent, or about 95percent, or about 96, 97, 98, or 99 percent identical to the nucleotidesequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleotidesequences encode a polypeptide that is about 85 percent, or about 90percent, or about 95 percent, or about 96, 97, 98, or 99 percentidentical to the polypeptide sequence as set forth in SEQ ID NO: 2 orSEQ ID NO: 4. Related nucleic acid molecules also include fragments ofthe above FGF-like nucleic acid molecules which are at least about 16contiguous nucleotides, or about 18, or about 20, or about 25, or about50, or about 75, or about 100, or greater than about 100 contiguousnucleotides. Related nucleic acid molecules also include fragments ofthe above FGF-like nucleic acid molecules which encode a polypeptide ofat least about 25 amino acid residues, or about 50, or about 75, orabout 100, or greater than about 100 amino acid residues.

Related nucleic acid molecules also include a nucleotide sequenceencoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO:4 with at least one conservative amino acid substitution and wherein thepolypeptide retains at least one FGF-like polypeptide activity, or anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 4 with at least one amino acid insertion and whereinthe polypeptide retains at least one FGF-like polypeptide activity, or anucleotide sequence encoding a polypeptide as set forth in either SEQ IDNO: 2 or SEQ ID NO: 4 with at least one amino acid deletion and whereinthe polypeptide retains at least one FGF-like polypeptide activity.Related nucleic acid molecules further include a nucleotide sequenceencoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO:4 which has a C- and/or N-terminal truncation and wherein thepolypeptide retains at least one FGF-like polypeptide activity. Relatednucleic acid molecules also include a nucleotide sequence encoding apolypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 4 withcombinations of modifications selected from the group consisting ofamino acid substitutions, amino acid insertions, amino acid deletions,C-terminal truncations, and N-terminal truncations and wherein thepolypeptide retains at least one FGF-like polypeptide activity.

Related FGF-like nucleic acid molecules include those molecules thatcomprise nucleotide sequences which hybridize under moderately or highlystringent conditions as defined herein with the complements of any ofthe above nucleic acid molecules. In preferred embodiments, the relatednucleic acid molecules comprise sequences which hybridize undermoderately or highly stringent conditions with the sequence as shown inSEQ ID NO: 1 or SEQ ID NO: 3, or with a molecule encoding a polypeptide,which polypeptide comprises the sequence as shown in SEQ ID NO: 2 or SEQID NO: 4, or with a nucleic acid fragment as defined above, or with anucleic acid fragment encoding a polypeptide as defined above. It isalso understood that related nucleic acid molecules include allelic orsplice variants of any of the above nucleic acids, and include sequenceswhich are complementary to any of the above nucleotide sequences.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that is free from at least one contaminatingnucleic acid molecule with which it is naturally associated, andpreferably substantially free from any other contaminating mammaliannucleic acid molecules which would interfere with its use in proteinproduction or its therapeutic or diagnostic use.

The term “allelic variant” refers to one of several possible naturallyoccurring alternate forms of a gene occupying a given locus on achromosome of an organism or a population of organisms.

The term “splice variant” refers to a nucleic acid molecule, usuallyRNA, which is generated by alternative processing of intron sequences inan RNA transcript.

The term “expression vector” refers to a vector that is suitable forpropagation in a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “highly stringency conditions” refers to those conditions that(1) employ low ionic strength reagents and high temperature for washing,for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ (SDS) at50° C., or (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin, 0.2% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphatebuffer (pH 6.5), 750 mM NaCl, and 75 mM sodium citrate at 42° C. Anotherexample is the use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and0.1% SDS.

The term “moderately stringency conditions” refers to conditions whichgenerally include the use of a washing solution and hybridizationconditions (e.g., temperature, ionic strength, and percentage of SDS)less stringent than described above. An example of moderately stringentconditions are conditions such as overnight incubation at 37° C. in asolution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μl/ml denatured sheared salmon sperm DNA,followed by washing in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

In certain preferred embodiments, where oligonucleotide probes are usedto screen cDNA or genomic libraries, high stringency conditions are usedwhich depend upon the melting temperature (T_(m)) of oligonucleotideprobes to target sequences. The T_(m) may be estimated using thefollowing formula (Bolton et al., Proc. Natl. Acad. Sci. U.S.A. 48:1390(1962)):

T _(m)=81.5−16.6(log [Na+])+0.41(% G+C)−(600/N)

wherein [Na+] is the sodium ion concentration in the hybridization (orwashing) solution;

% G+C is guanine and cytosine content in the oligonucleotide probe; and

N is the probe length in nucleotides.

An example of a high stringency solution is 6×SSC and 0.05% sodiumpyrophosphate at a temperature of 35-63° C., depending on the length ofthe oligonucleotide probe. For example, according to certainembodiments, 14 base pair probes are washed at 35-40° C., 17 base pairprobes are washed at 45-50° C., 20 base pair probes are washed at 52-57°C., and 23 base pair probes are washed at 57-63° C. The temperature canbe increased 2-3° C. where the background non-specific binding appearshigh. A second high stringency solution utilizes tetramethylammoniumchloride (TMAC) for washing oligonucleotide probes. One stringentwashing solution is 3 M TMAC, 50 mM Tris-HCl, pH 8.0, and 0.2% SDS. Thewashing temperature using this solution is a function of the length ofthe probe. For example, 14 base pair probes are washed at 35-40° C., 17base pair probes are washed at about 45-50° C., 20 base pair probes arewashed at 52-57° C., and 23 base pair probes are washed at 57-63° C.

The term “FGF-like polypeptides” refers to a polypeptide comprising theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and relatedpolypeptides described herein. Related polypeptides include: allelicvariants; splice variants; fragments; derivatives; substitution,deletion, and insertion variants; fusion polypeptides; and orthologs.FGF-like polypeptides may be mature polypeptides, as defined herein, andmay or may not have an amino terminal methionine residue, depending onthe method by which they are prepared.

The term “FGF-like polypeptide fragment” refers to a peptide orpolypeptide that comprises less than the full length amino acid sequenceof an FGF-like polypeptide as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.Such a fragment may arise, for example, from a truncation at the aminoterminus, a truncation at the carboxyl terminus, and/or an internaldeletion of a residue(s) from the amino acid sequence. FGF-likefragments may result from alternative RNA splicing or from in vivoprotease activity.

The term “FGF-like polypeptide variants” refers to FGF-like polypeptidescomprising amino acid sequences which contain one or more amino acidsequence substitutions, deletions, and/or additions as compared to theFGF-like polypeptide amino acid sequence set forth in SEQ ID NO: 2 orSEQ ID NO: 4. Variants may be naturally occurring or artificiallyconstructed. Such FGF-like polypeptide variants may be prepared from thecorresponding nucleic acid molecules encoding said variants, which havea DNA sequence that varies accordingly from the DNA sequences for wildtype FGF-like polypeptides as set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

The term “FGF-like fusion polypeptide” refers to a fusion of an FGF-likepolypeptide, fragment, variant, or derivative thereof, with aheterologous peptide or polypeptide.

The term “FGF-like polypeptide derivatives” refers to FGF-likepolypeptides, variants, or fragments thereof, that have been chemicallymodified, as for example, by covalent attachment of one or morepolymers, including, but not limited to, water soluble polymers,N-linked or O-linked carbohydrates, sugars, phosphates, and/or othersuch molecules. The derivatives are modified in a manner that isdifferent from naturally occurring FGF-like polypeptide, either in thetype or location of the molecules attached to the polypeptide.Derivatives further include the deletion of one or more chemical groupsnaturally attached to the FGF-like polypeptide.

The terms “biologically active FGF-like polypeptides,” “biologicallyactive FGF-like polypeptide fragments,” “biologically active FGF-likepolypeptide variants,” and “biologically active FGF-like polypeptidederivatives” refer to FGF-like polypeptides having at least one activitycharacteristic of an FGF-like polypeptide, such as stimulation of cellswithin or near the liver, regulation of intestinal cell activity,stimulation of cells within or near pancreatic islets, regulation ofneuronal cells, stimulation or inhibition of angiogenesis, stimulationof epithelium or mesenchymal components of granulation tissue,stimulation of corneal epithelium, lens, or retinal tissue, regenerationof renal tubules, hematopoietic cell regulation, regulation of hairfollicle growth, regulation of pulmonary epithelium, or stimulation ofeither epithelial, mesenchymal, hematopoietic, or neuronal cells ortissues. In general, FGF-like polypeptides, and variants, fragments andderivatives thereof, will have at least one activity characteristic ofan FGF-like polypeptide such as those activities listed above. Inaddition, an FGF-like polypeptide may be active as an immunogen (i.e.,the polypeptide contains at least one epitope to which antibodies may beraised).

“Naturally occurring” when used in connection with biological materialssuch as nucleic acid molecules, polypeptides, host cells, and the like,refers to that which are found in nature and not manipulated by a humanbeing.

The term “isolated polypeptide” refers to a polypeptide of the inventionthat is free from at least one contaminating polypeptide that is foundin its natural environment, and preferably substantially free from anyother contaminating mammalian polypeptides which would interfere withits therapeutic or diagnostic use.

The term “ortholog” refers to a polypeptide that corresponds to apolypeptide identified from a different species. For example, murine andhuman FGF-like polypeptides are considered orthologs of one another.

The term “mature FGF-like polypeptide” refers to a polypeptide lacking aleader sequence and may also include other modifications of apolypeptide such as proteolytic processing of the amino terminus (withor without a leader sequence) and/or the carboxyl terminus, cleavage ofa smaller polypeptide from a larger precursor, N-linked and/or O-linkedglycosylation, and other post-translational modifications understood bythose with skill in the art.

The terms “effective amount” and “therapeutically effective amount”refer to the amount of a FGF-like polypeptide that is useful ornecessary to support an observable level of one or more biologicalactivities of the FGF-like polypeptides as set forth above.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween polypeptide or nucleic acid molecule sequences, as the case maybe, as determined by the match between strings of nucleotide or aminoacid sequences. “Identity” measures the percent of identical matchesbetween two or more sequences with gap alignments addressed by aparticular mathematical model of computer programs (i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity,” refers to a measure of similarity which includes bothidentical matches and conservative substitution matches. Sinceconservative substitutions apply to polypeptides and not nucleic acidmolecules, similarity only deals with polypeptide sequence comparisons.If two polypeptide sequences have, for example, 10 out of 20 identicalamino acids, and the remainder are all non-conservative substitutions,then the percent identity and similarity would both be 50%. If in thesame example, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15 out of 20). Therefore, in cases where thereare conservative substitutions, the degree of similarity between twopolypeptide sequences will be higher than the percent identity betweenthose two sequences.

The term “conservative amino acid substitution” refers to a substitutionof a native amino acid residue with a normative residue such that thereis little or no effect on the polarity or charge of the amino acidresidue at that position. For example, a conservative substitutionresults from the replacement of a non-polar residue in a polypeptidewith any other non-polar residue. Furthermore, any native residue in thepolypeptide may also be substituted with alanine, as has been previouslydescribed for “alanine scanning mutagenesis” (Cunnigham et al., Science244:1081-85 (1989)). General rules for conservative amino acidsubstitutions are set forth in Table I.

TABLE I Conservative Amino Acid Substitutions Exemplary PreferredOriginal Residues Substitutions Substitutions Ala Val, Leu, Ile Val ArgLys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser GlnAsn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu,Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met,Ala, Phe Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile,Ala, Leu Tyr Pro Ala Ala Ser Thr Thr Thr Ser Ser Trp Tyr, Phe Tyr TyrTrp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, NorleucineConservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues that are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties.

Conservative modifications to the amino acid sequence (and thecorresponding modifications to the encoding nucleotides) are expected toproduce FGF-like polypeptide having functional and chemicalcharacteristics similar to those of naturally occurring FGF-likepolypeptide. In contrast, substantial modifications in the functionaland/or chemical characteristics of FGF-like polypeptide may beaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. Naturally occurringresidues may be divided into groups based on common side chainproperties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human FGF-like moleculethat are homologous with non-human FGF-like polypeptide, or into thenon-homologous regions of the molecule.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods, including butnot limited to those described in Computational Molecular Biology (A. M.Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics andGenome Projects (D. W. Smith, ed., Academic Press 1993); ComputerAnalysis of Sequence Data (Part 1, A. M. Griffin and H. G. Griffin,eds., Humana Press 1994); G. von Heinle, Sequence Analysis in MolecularBiology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov andJ. Devereux, eds., M. Stockton Press 1991); and Carillo et al., SIAM J.Applied Math. 48:1073 (1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nuc. Acids Res. 12:387 (1984); Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Atschul et al., J.Mol. Biol. 215:403-10 (1990)). The BLAST X program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda, Md.);Altschul et al., 1990, supra). The well-known Smith Waterman algorithmmay also be used to determine identity.

By way of example, using the computer algorithm GAP (Genetics ComputerGroup), two polypeptides for which the percent sequence identity is tobe determined are aligned for optimal matching of their respective aminoacids (the “matched span,” as determined by the algorithm). A gapopening penalty (which is calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 0.1× the gap opening penalty),as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used inconjunction with the algorithm. A standard comparison matrix (seeDayhoff et al., 5 Atlas of Protein Sequence and Structure (Supp. 3 1978)for the PAM250 comparison matrix; see Henikoff et al., Proc. Natl. Acad.Sci USA 89:10915-19 (1992) for the BLOSUM 62 comparison matrix) is alsoused by the algorithm.

Preferred parameters for polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-53 (1970)

Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl. Acad.Sci. U.S.A. 89:10915-19 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisoninclude the following:

Algorithm: Needleman et al., J. Mol Biol. 48:443-53 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version 9, September, 1997. Theparticular choices to be made will depend on the specific comparison tobe made, such as DNA to DNA, protein to protein, protein to DNA; andadditionally, whether the comparison is between given pairs of sequences(in which case GAP or BestFit are generally preferred) or between onesequence and a large database of sequences (in which case FASTA orBLASTA are preferred).

Sequence analysis of an isolated mouse cDNA (murine FGF-like protein;SEQ ID NO: 3) indicated that it encoded a novel member of the FGF familyof proteins. The murine FGF-like gene comprises a 630 bp open readingframe encoding a protein of 210 amino acids (FIG. 1). The murinesequence was used to identify the human FGF-like ortholog. Sequenceanalysis of four human FGF-like polypeptide cDNA clones indicated thatthe human FGF-like gene comprises a 627 bp open reading frame encoding aprotein of 209 amino acids (FIGS. 2A-2B).

FIGS. 3A-3D illustrate the amino acid sequence alignment of humanFGF-like protein, murine FGF-like protein, and other members of the FGFfamily. Computer analysis of the predicted murine FGF-like polypeptide,using the FASTA program of the Swissprot database, indicated that theprotein was most closely related to murine FGF-6, FGF-15, and FGF-4.Using the GAP program, murine FGF-like polypeptide was found to be 32%identical to murine FGF-6 and 28% identical to murine FGF-4. Computeranalysis also indicated that the murine FGF-like polypeptide, similar toFGF-6, FGF-4, and FGF-15 but in contrast to FGF-1 and FGF-2, possessed apotential signal peptide at its amino terminus. The murine FGF-likepolypeptide is 79% identical to the human FGF-like protein.

Nucleic Acid Molecules

Recombinant DNA methods used herein are generally those set forth inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) and/or Current Protocols in MolecularBiology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons1994).

The invention provides for nucleic acid molecules as described hereinand methods for obtaining the molecules. A gene or cDNA encoding anFGF-like polypeptide or fragment thereof may be obtained byhybridization screening of a genomic or cDNA library, or by PCRamplification. Probes or primers useful for screening a library byhybridization can be generated based on sequence information for otherknown genes or gene fragments from the same or a related family ofgenes, such as, for example, conserved motifs. In addition, where a geneencoding FGF-like polypeptide has been identified from one species, allor a portion of that gene may be used as a probe to identifycorresponding genes from other species (orthologs) or related genes fromthe same species (homologs). The probes or primers may be used to screencDNA libraries from various tissue sources believed to express theFGF-like gene. In addition, part or all of a nucleic acid moleculehaving the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 may beused to screen a genomic library to identify and isolate a gene encodingFGF-like polypeptide. Typically, conditions of moderate or highstringency will be employed for screening to minimize the number offalse positives obtained from the screen.

Nucleic acid molecules encoding FGF-like polypeptides may also beidentified by expression cloning which employs detection of positiveclones based upon a property of the expressed protein. Typically,nucleic acid libraries are screened by binding an antibody or otherbinding partner (e.g., receptor or ligand) to cloned proteins which areexpressed and displayed on the host cell surface. The antibody orbinding partner is modified with a detectable label to identify thosecells expressing the desired clone.

Another means of preparing a nucleic acid molecule encoding an FGF-likepolypeptide or fragment thereof is chemical synthesis using methods wellknown to the skilled artisan such as those described by Engels et al.,Angew. Chem. Intl. Ed. 28:716-34 (1989). These methods include, interalfa, the phosphotriester, phosphoramidite, and FI-phosphonate methodsfor nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry. Typically, the DNA encoding the FGF-like polypeptide will beseveral hundred nucleotides in length. Nucleic acids larger than about100 nucleotides can be synthesized as several fragments using thesemethods. The fragments can then be ligated together to form thefull-length FGF-like polypeptide. Usually, the DNA fragment encoding theamino terminus of the polypeptide will have an ATG, which encodes amethionine residue. This methionine may or may not be present on themature form of the FGF-like polypeptide, depending on whether thepolypeptide produced in the host cell is designed to be secreted fromthat cell.

In some cases, it may be desirable to prepare nucleic acid moleculesencoding FGF-like polypeptide variants. Nucleic acid molecules encodingvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for optimal expression of an FGF-like polypeptide in agiven host cell. Particular codon alterations will depend upon theFGF-like polypeptide and host cell selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Ecohigh._Cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.Other useful codon frequency tables include “Celegans_high.cod,”“Celegans_low.cod,” “Drosophila_high.cod,” “Human_high.cod,”“Maize_high.cod,” and “Yeasthigh.cod.”

In other embodiments, nucleic acid molecules encode FGF-like variantswith conservative amino acid substitutions as defined above, FGF-likevariants comprising an addition and/or a deletion of one or moreN-linked or O-linked glycosylation sites, FGF-like variants havingdeletions and/or substitutions of one or more cysteine residues, orFGF-like polypeptide fragments as described above. In addition, nucleicacid molecules may encode any combination of FGF-like variants,fragments, and fusion polypeptides described herein.

Vectors and Host Cells

A nucleic acid molecule encoding an FGF-like polypeptide is insertedinto an appropriate expression vector using standard ligationtechniques. The vector is typically selected to be functional in theparticular host cell employed (i.e., the vector is compatible with thehost cell machinery such that amplification of the gene and/orexpression of the gene can occur). A nucleic acid molecule encoding anFGF-like polypeptide may be amplified/expressed in prokaryotic, yeast,insect (baculovirus systems) and/or eukaryotic host cells. Selection ofthe host cell will depend in part on whether an FGF-like polypeptide isto be post-translationally modified (e.g., glycosylated and/orphosphorylated). If so, yeast, insect, or mammalian host cells arepreferable. For a review of expression vectors, see 185 Meth. Enz. (D.V. Goeddel, ed., Academic Press 1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotides: a promoter, one or moreenhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a leader sequence for secretion, a ribosomebinding site, a polyadenylation sequence, a polylinker region forinserting the nucleic acid encoding the polypeptide to be expressed, anda selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may contain a “tag” sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the FGF-likepolypeptide coding sequence; the oligonucleotide molecule encodespolyHis (such as hexaHis), or other “tag” such as FLAG, HA (hemaglutininInfluenza virus) or myc for which commercially available antibodiesexist. This tag is typically fused to the polypeptide upon expression ofthe polypeptide, and can serve as a means for affinity purification ofthe FGF-like polypeptide from the host cell. Affinity purification canbe accomplished, for example, by column chromatography using antibodiesagainst the tag as an affinity matrix. Optionally, the tag cansubsequently be removed from the purified FGF-like polypeptide byvarious means such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), or synthetic, or nativesequences which normally function to regulate FGF-like expression. Assuch, the source of flanking sequences may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequences is functional in, and can beactivated by, the host cell machinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein other than the FGF-like gene flankingsequences will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of one or more flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described above for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species.

Where the flanking sequence is not known, a fragment of DNA containing aflanking sequence may be isolated from a larger piece of DNA that maycontain, for example, a coding sequence or even another gene or genes.Isolation may be accomplished by restriction endonuclease digestion toproduce the proper DNA fragment followed by isolation using agarose gelpurification, Qiagen® (Valencia, Calif.) column chromatography, or othermethods known to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

An origin of replication is typically a part of prokaryotic expressionvectors purchased commercially, and aids in the amplification of thevector in a host cell. Amplification of the vector to a certain copynumber can, in some cases, be important for optimal expression of theFGF-like polypeptide. If the vector of choice does not contain an originof replication site, one may be chemically synthesized based on a knownsequence, and ligated into the vector. The origin of replication fromthe plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly,Mass.) is suitable for most Gram-negative bacteria and various origins(e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) orpapillomaviruses such as HPV or BPV) are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (for example, the SV40 origin isoften used only because it contains the early promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding regions and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, for example, ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue of themarker present in the vector. Selection pressure is imposed by culturingthe transformed cells under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesFGF-like polypeptide. As a result, increased quantities of FGF-likepolypeptide are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the FGF-like polypeptideto be expressed. The Shine-Dalgarno sequence is varied but is typicallya polypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth above and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct an FGF-likepolypeptide out of the host cell. Typically, the signal sequence ispositioned in the coding region of the FGF-like nucleic acid molecule,or directly at the 5′ end of the FGF-like polypeptide coding region.Many signal sequences have been identified, and any of them that arefunctional in the selected host cell may be used in conjunction with theFGF-like gene or cDNA. Therefore, a signal sequence may be homologous(naturally occurring) or heterologous to the FGF-like gene or cDNA,Additionally, a signal sequence may be chemically synthesized usingmethods set forth above. In most cases, secretion of an FGF-likepolypeptide from the host cell via the presence of a signal peptide willresult in the removal of the signal peptide from the FGF-likepolypeptide. The signal sequence may be a component of the vector, or itmay be a part of FGF-like DNA that is inserted into the vector.

Included within the scope of this invention is the native FGF-likesignal sequence joined to an FGF-like coding region and a heterologoussignal sequence joined to an FGF-like coding region. The heterologoussignal sequence selected should be one that is recognized and processed,i.e., cleaved by a signal peptidase, by the host cell. For prokaryotichost cells that do not recognize and process the native FGF-like signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, or heat-stable enterotoxin II leaders. Foryeast secretion, the native FGF-like signal sequence may be substitutedby the yeast invertase, alpha factor, or acid phosphatase leaders. Inmammalian cell expression the native signal sequence is satisfactory,although other mammalian signal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid found in thepeptidase cleavage site, attached to the N-terminus. Alternatively, useof some enzyme cleavage sites may result in a slightly truncated form ofthe desired FGF-like polypeptide, if the enzyme cuts at such area withinthe mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the FGF-like gene especially where the gene used is afull-length genomic sequence or a fragment thereof. Where the intron isnot naturally occurring within the gene (as for most cDNAs), the intronmay be obtained from another source. The position of the intron withrespect to flanking sequences and the FGF-like gene is generallyimportant, as the intron must be transcribed to be effective. Thus, whenan FGF-like cDNA molecule is being expressed, the preferred position forthe intron is 3′ to the transcription start site and 5′ to the poly-Atranscription termination sequence. Preferably, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt the coding sequence. Any intron from anysource, including any viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this invention, provided thatit is compatible with the host cell into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the FGF-like protein. Promotersare untranslated sequences located upstream (i.e., 5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of the structural gene.Promoters are conventionally grouped into one of two classes: induciblepromoters and constitutive promoters. Inducible promoters initiateincreased levels of transcription from DNA under their control inresponse to some change in culture conditions, such as the presence orabsence of a nutrient or a change in temperature. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. These promoters are operably linked to the DNA encoding FGF-likepolypeptide by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence into thevector. The native FGF-like promoter sequence may be used to directamplification and/or expression of FGF-like DNA. A heterologous promoteris preferred, however, if it permits greater transcription and higheryields of the expressed protein as compared to the native promoter, andif it is compatible with the host cell system that has been selected foruse.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase, atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence, using linkers or adapters asneeded to supply any required restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude those obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40). Othersuitable mammalian promoters include heterologous mammalian promoters,for example, heat-shock promoters and the actin promoter.

Additional promoters which may be of interest in controlling FGF-likegene expression include, but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, Nature 290:304-10 (1981)); the CMVpromoter; the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto, et al., Cell 22:787-97 (1980)); the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1444-45 (1981)); the regulatory sequences of the metallothionine gene(Brinster et al., Nature 296:39-42 (1982)); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,Proc. Natl. Acad. Sci. U.S.A., 75:3727-31 (1978)); or the tac promoter(DeBoer et al., Proc. Natl. Acad. Sci. U.S.A., 80:21-25 (1983)). Also ofinterest are the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:the elastase I gene control region which is active in pancreatic acinarcells (Swift et al., Cell 38:639-46 (1984); Ornitz et al., Cold SpringHarbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology7:425-515 (1987)); the insulin gene control region which is active inpancreatic beta cells (Hanahan, Nature 315:115-22 (1985)); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., Cell 38:647-58 (1984); Adames et al., Nature318:533-38 (1985); Alexander et al., Mol. Cell. Biol., 7:1436-44(1987)); the mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-95 (1986)); the albumin gene control region which is active inliver (Pinkert et al., Genes and Devel. 1:268-76 (1987)); thealpha-feto-protein gene control region which is active in liver(Krumlauf et al., Mol. Cell. Biol., 5:1639-48 (1985); Hammer et al.,Science 235:53-58 (1987)); the alpha 1-antitrypsin gene control regionwhich is active in the liver (Kelsey et al., Genes and Devel. 1:161-71,1987)); the beta-globin gene control region which is active in myeloidcells (Mogram et al., Nature 315:338-40 (1985); Kollias et al., Cell46:89-94 (1986)); the myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., Cell48:703-12 (1987)); the myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, Nature 314:283-86 (1985)); and thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science 234:1372-78 (1986)).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding an FGF-like protein of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease its transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation of eukaryotic promoters. While an enhancer may be splicedinto the vector at a position 5′ or 3′ to FGF-like DNA, it is typicallylocated at a site 5′ from the promoter.

Expression vectors of the invention may be constructed from startingvectors such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences set forth above are not already present in thevector to be used, they may be individually obtained and ligated intothe vector. Methods used for obtaining each of the flanking sequencesare well known to one skilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen, SanDiego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacH, Invitrogen), pDSR-alpha(PCT Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island,N.Y.).

Additional possible vectors include, but are not limited to, cosmids,plasmids, or modified viruses, but the vector system must be compatiblewith the selected host cell. Such vectors include, but are not limitedto plasmids such as Bluescript® plasmid derivatives (a high copy numberColE1-based phagemid, Stratagene Cloning Systems, La Jolla Calif.), PCRcloning plasmids designed for cloning Taq-amplified PCR products (e.g.,TOPO™ TA Cloning® Kit, PCR2.1® plasmid derivatives, Invitrogen,Carlsbad, Calif.), and mammalian, yeast or virus vectors such as abaculovirus expression system (pBacPAK plasmid derivatives, Clontech,Palo Alto, Calif.). The recombinant molecules can be introduced intohost cells via transformation, transfection, infection, electroporation,or other known techniques.

After the vector has been constructed and a nucleic acid moleculeencoding an FGF-like polypeptide has been inserted into the proper siteof the vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, synthesizesan FGF-like polypeptide which can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted).Selection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity, such as glycosylation orphosphorylation, and ease of folding into a biologically activemolecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Examples include mammalian cells, such as Chinese hamster ovarycells (CHO), CHO DHFR− cells (Urlaub et al., Proc. Natl. Acad. Sci.U.S.A. 97:4216-20 (1980)), human embryonic kidney (HEK) 293 or 293Tcells, or 3T3 cells. The selection of suitable mammalian host cells andmethods for transformation, culture, amplification, screening, productproduction and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 and COS-7 cell lines, and theCV-1 cell line. Further exemplary mammalian host cells include primatecell lines and rodent cell lines, including transformed cell lines.Normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, as well as primary explants, are also suitable.Candidate cells may be genotypically deficient in the selection gene, ormay contain a dominantly acting selection gene. Other suitable mammaliancell lines include but are not limited to, mouse neuroblastoma N2Acells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c orNH-1 mice, BHK or HaK hamster cell lines. Each of these cell lines isknown by and available to those skilled in the art of proteinexpression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5, DH10, and MC1061) are well known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Preferred yeast cells include, for example,Saccharomyces cerivisae.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described, forexample, in Kitts et al., Biotechniques, 14:810-17 (1993); Lucklow,Curr. Opin. Biotechnol. 4:564-72 (1993); and Lucklow et al., J. Virol.,67:4566-79 (1993). Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

Transformation or transfection of an expression vector for an FGF-likepolypeptide into a selected host cell may be accomplished by well knownmethods including methods such as calcium chloride, electroporation,microinjection, lipofection or the DEAE-dextran method. The methodselected will in part be a function of the type of host cell to be used.These methods and other suitable methods are well known to the skilledartisan, and are set forth, for example, in Sambrook et al., supra.

One may also use transgenic animals to express glycosylated FGF-likepolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produceFGF-like polypeptides, however, in general, the glycosylation occurringin plants is different from that produced in mammalian cells, and mayresult in a glycosylated product which is not suitable for humantherapeutic use.

Polypeptide Production

Host cells comprising an FGF-like polypeptide expression vector (i.e.,transformed or transfected) may be cultured using standard media wellknown to the skilled artisan. The media will usually contain allnutrients necessary for the growth and survival of the cells. Suitablemedia for culturing E. coli cells are for example, Luria Broth (LB)and/or Terrific Broth (TB). Suitable media for culturing eukaryoticcells are RPMI 1640, MEM, DMEM, all of which may be supplemented withserum and/or growth factors as required by the particular cell linebeing cultured. A suitable medium for insect cultures is Grace's mediumsupplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calfserum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transfected or transformed cells is added as a supplement to themedia. The compound to be used will be dictated by the selectable markerelement present on the plasmid with which the host cell was transformed.For example, where the selectable marker element is kanamycinresistance, the compound added to the culture medium will be kanamycin.Other compounds for selective growth include ampicillin, tetracycline,and neomycin.

The amount of an FGF-like polypeptide produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If an FGF-like polypeptide has been designed to be secreted from thehost cells, the majority of polypeptide may be found in the cell culturemedium. If however, the FGF-like polypeptide is not secreted from thehost cells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram-negative bacteriahost cells).

For an FGF-like polypeptide situated in the host cell cytoplasm and/ornucleus, the host cells are typically first disrupted mechanically orwith detergent to release the intra-cellular contents into a bufferedsolution. FGF-like polypeptide can then be isolated from this solution.

Purification of an FGF-like polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (FGF-likepolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen) at either its carboxyl oramino terminus, it may essentially be purified in a one-step process bypassing the solution through an affinity column where the column matrixhas a high affinity for the tag or for the polypeptide directly (i.e., amonoclonal antibody specifically recognizing FGF-like polypeptide). Forexample, polyhistidine binds with great affinity and specificity tonickel and thus an affinity column of nickel (such as the Qiagen® nickelcolumns) can be used for purification of FGF-like polypeptide/polyHis.See, e.g., Current Protocols in Molecular Biology §10.11.8 (Ausubel etal., eds., John Wiley & Sons 1993).

Where an FGF-like polypeptide is prepared without a tag attached, and noantibodies are available, other well-known procedures for purificationcan be used. Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific).In some cases, two or more of these techniques may be combined toachieve increased purity.

If an FGF-like polypeptide is produced intracellularly, theintracellular material (including inclusion bodies for gram-negativebacteria) can be extracted from the host cell using any standardtechnique known to the skilled artisan. For example, the host cells canbe lysed to release the contents of the periplasm/cytoplasm by Frenchpress, homogenization, and/or sonication followed by centrifugation.

If an FGF-like polypeptide has formed inclusion bodies in the cytosol,the inclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The FGF-like polypeptide in its now soluble form canthen be analyzed using gel electrophoresis, immunoprecipitation, or thelike. If it is desired to isolate the FGF-like polypeptide, isolationmay be accomplished using standard methods such as those set forth belowand in Marston et al., Meth. Enz., 182:264-75 (1990).

In some cases, an FGF-like polypeptide may not be biologically activeupon isolation. Various methods for “refolding” or converting thepolypeptide to its tertiary structure and generating disulfide linkagescan be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridges. Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolvent maybe used or may be needed to increase the efficiency of the refolding andthe more common reagents used for this purpose include glycerol,polyethylene glycol of various molecular weights, arginine and the like.

If inclusion bodies are not formed to a significant degree uponexpression of an FGF-like polypeptide, the polypeptide will be foundprimarily in the supernatant after centrifugation of the cell homogenateand may be further isolated from the supernatant using methods such asthose set forth below.

In situations where it is preferable to partially or completely purifyan FGF-like polypeptide such that it is partially or substantially freeof contaminants, standard methods known to the one skilled in the artmay be used. Such methods include, without limitation, separation byelectrophoresis followed by electroelution, various types ofchromatography (affinity, immunoaffinity, molecular sieve, and/or ionexchange), and/or high pressure liquid chromatography. In some cases, itmay be preferable to use more than one of these methods for completepurification.

FGF-like polypeptides, fragments, and/or derivatives thereof may also beprepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art such as those set forth byMerrifield et al., J. Am. Chem. Soc. 85:2149 (1963); Houghten et al.,Proc Natl Acad. Sci. USA 82:5132 (1985); and Stewart and Young, SolidPhase Peptide Synthesis (Pierce Chemical Co. 1984). Such polypeptidesmay be synthesized with or without a methionine on the amino terminus.Chemically synthesized FGF-like polypeptides or fragments may beoxidized using methods set forth in these references to form disulfidebridges. FGF-like polypeptides, fragments or derivatives are expected tohave comparable biological activity to the corresponding FGF-likepolypeptides, fragments or derivatives produced recombinantly orpurified from natural sources, and thus may be used interchangeably withrecombinant or natural FGF-like polypeptide.

Another means of obtaining FGF-like polypeptide is via purification frombiological samples such as source tissues and/or fluids in which theFGF-like polypeptide is naturally found. Such purification can beconducted using methods for protein purification as described above. Thepresence of the FGF-like polypeptide during purification may bemonitored using, for example, an antibody prepared against recombinantlyproduced FGF-like polypeptide or peptide fragments thereof.

Polypeptides

Polypeptides of the invention include isolated FGF-like polypeptides andpolypeptides related thereto including fragments, variants, fusionpolypeptides, and derivatives as defined hereinabove.

FGF-like polypeptide fragments of the invention may result fromtruncations at the amino terminus (with or without a leader sequence),truncations at the carboxyl terminus, and/or deletions internal to thepolypeptide. In preferred embodiments, truncations and/or deletionscomprise about 10 amino acids, or about 20 amino acid, or about 50 aminoacids, or about 75 amino acids, or about 100 amino acids, or more thanabout 100 amino acids. The polypeptide fragments so produced willcomprise about 25 contiguous amino acids, or about 50 amino acids, orabout 75 amino acids, or about 100 amino acids, or about 150 aminoacids, or about 200 amino acids. Such FGF-like polypeptide fragments mayoptionally comprise an amino terminal methionine residue.

FGF-like polypeptide variants of the invention include one or more aminoacid substitutions, additions and/or deletions as compared to SEQ ID NO:2 or SEQ ID NO: 4. In preferred embodiments, the variants have from 1 to3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, orfrom 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, ormore than 100 amino acid substitutions, insertions, additions and/ordeletions, wherein the substitutions may be conservative, as definedabove, non-conservative, or any combination thereof, and wherein theFGF-like polypeptide variant retains an FGF-like activity. The variantsmay have additions of amino acid residues either at the carboxylterminus or at the amino terminus (with or without a leader sequence).

Preferred FGF-like polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites has been alteredcompared to native FGF-like polypeptide. In one embodiment, FGF-likevariants comprise a greater or a lesser number of N-linked glycosylationsites. An N-linked glycosylation site is characterized by the sequence:Asn-X-Ser or Thr, where the amino acid residue designated as “X” may beany type of amino acid except proline. Substitution(s) of amino acidresidues to create this sequence provides a potential new site foraddition of an N-linked carbohydrate chain. Alternatively, substitutionsto eliminate this sequence will remove an existing N-linked carbohydratechain. Also provided is a rearrangement of N-linked carbohydrate chainswherein one or more N-linked glycosylation sites (typically those thatare naturally occurring) are eliminated and one or more new N-linkedsites are created. Additional preferred FGF-like variants includecysteine variants, wherein one or more cysteine residues are deleted orsubstituted with another amino acid (e.g., serine). Cysteine variantsare useful when FGF-like polypeptide must be refolded into abiologically active conformation such as after isolation of insolubleinclusive bodies. Cysteine variants generally have fewer cysteineresidues than the native protein, and typically have an even number tominimize interactions resulting from unpaired cysteines.

One skilled in the art will be able to determine suitable variants ofthe native FGF-like polypeptide using well-known techniques. Forexample, one may be able to predict suitable areas of the molecule thatmay be changed without destroying biological activity. Also, one skilledin the art will realize that even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

For predicting suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of FGF-like polypeptide to such similar polypeptides. Aftermaking such a comparison, one skilled in the art would be able todetermine residues and portions of the molecules that are conservedamong similar polypeptides. One skilled in the art would know thatchanges in areas of the FGF-like molecule that are not conserved wouldbe less likely to adversely affect biological activity and/or structure.One skilled in the art would also know that, even in relativelyconserved regions, one could have likely substituted chemically similaramino acids for the naturally occurring residues is while retainingactivity (conservative amino acid residue substitutions).

Also, one skilled in the art may review structure-function studiesidentifying residues in similar polypeptides that are important foractivity or structure. In view of such a comparison, one skilled in theart can predict the importance of amino acid residues in FGF-likepolypeptide that correspond to amino acid residues that are importantfor activity or structure in similar polypeptides. One skilled in theart may opt for chemically similar amino acid substitutions for suchpredicted important amino acid residues of FGF-like polypeptide.

If available, one skilled in the art can also analyze thethree-dimensional structure and amino acid sequence in relation to thatstructure in similar polypeptides. In view of that information, oneskilled in the art may be able to predict the alignment of amino acidresidues of FGF-like polypeptide with respect to its three dimensionalstructure. One skilled in the art may choose not to make radical changesto amino acid residues predicted to be on the surface of the protein,since such residues may be involved in important interactions with othermolecules.

Moreover, one skilled in the art could generate test variants containinga single amino acid substitution at each amino acid residue. Thevariants could be screened using activity assays disclosed in thisapplication. Such variants could be used to gather information aboutsuitable variants. For example, if one discovered that a change to aparticular amino acid residue resulted in destroyed activity, variantswith such a change would be avoided. In other words, based oninformation gathered from such experiments, when attempting to findadditional acceptable variants, one skilled in the art would have knownthe amino acids where further substitutions should be avoided eitheralone or in combination with other mutations.

FGF-like fusion polypeptides of the invention comprise FGF-likepolypeptides, fragments, variants, or derivatives fused to aheterologous peptide or protein. Heterologous peptides and proteinsinclude, but are not limited to: an epitope to allow for detectionand/or isolation of an FGF-like fusion polypeptide; a transmembranereceptor protein or a portion thereof, such as an extracellular domain,or a transmembrane and intracellular domain; a ligand or a portionthereof which binds to a transmembrane receptor protein; an enzyme orportion thereof which is catalytically active; a protein or peptidewhich promotes oligomerization, such as leucine zipper domain; and aprotein or peptide which increases stability, such as an immunoglobulinconstant region. An FGF-like polypeptide may be fused to itself or to afragment, variant, or derivative thereof. Fusions may be made either atthe amino terminus or at the carboxyl terminus of an FGF-likepolypeptide, and may be direct with no linker or adapter molecule or maybe through a linker or adapter molecule, such as one or more amino acidresidues up to about 20 amino acids residues, or up to about 50 aminoacid residues. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for a protease toallow for separation of the fused moieties.

In a further embodiment of the invention, an FGF-like polypeptide,fragment, variant and/or derivative is fused to an Fc region of humanIgG. In one example, a human IgG hinge, CH2 and CH3 region may be fusedat either the N-terminus or C-terminus of the FGF-like polypeptidesusing methods known to the skilled artisan. In another example, aportion of a hinge regions and CH2 and CH3 regions may be fused. TheFGF-like Fc-fusion polypeptide so produced may be purified by use of aProtein A affinity column. In addition, peptides and proteins fused toan Fc region have been found to exhibit a substantially greaterhalf-life in vivo than the unfused counterpart. Also, a fusion to an Fcregion allows for dimerization/multimerization of the fusionpolypeptide. The Fc region may be a naturally occurring Fc region, ormay be altered to improve certain qualities, such as therapeuticqualities, circulation time, reduced aggregation, etc.

FGF-like polypeptide derivatives are included in the scope of thepresent invention. Such derivatives are chemically modified FGF-likepolypeptide compositions in which FGF-like polypeptide is linked to apolymer. The polymer selected is typically water-soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer may be ofany molecular weight, and may be branched or unbranched. Included withinthe scope of FGF-like polypeptide polymers is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose, or other carbohydratebased polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol,propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinylalcohol. Also encompassed by the invention are bifunctional PEGcross-linking molecules that may be used to prepare covalently attachedFGF-like polypeptide multimers

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A reactivealdehyde is, for example, polyethylene glycol propionaldehyde, which iswater stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (seeU.S. Pat. No. 5,252,714).

Pegylation of FGF-like polypeptides may be carried out by any of thepegylation reactions known in the art, as described for example in thefollowing references: Francis et al., Focus on Growth Factors 3, 4-10(1992); EP 0 154 316; EP 0 401 384 and U.S. Pat. No. 4,179,337.Pegylation may be carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule (or an analogousreactive water-soluble polymer) as described below.

One water-soluble polymer for use herein is polyethylene glycol,abbreviated PEG. As used herein, polyethylene glycol is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethyleneglycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated FGF-likepolypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby FGF-likepolypeptide becomes attached to one or more PEG groups, and (b)obtaining the reaction product(s). In general, the optimal reactionconditions for the acylation reactions will be determined based on knownparameters and the desired result. For example, the larger the ratio ofPEG: protein, the greater the percentage of poly-pegylated product.

In a preferred embodiment, the FGF-like polypeptide derivative will havea single PEG moiety at the amino terminus. See U.S. Pat. No. 5,234,784,herein incorporated by reference.

Generally, conditions that may be alleviated or modulated byadministration of the present FGF-like polypeptide derivative includethose described herein for FGF-like polypeptides. However, the FGF-likepolypeptide derivative disclosed herein may have additional activities,enhanced or reduced biological activity, or other characteristics, suchas increased or decreased half-life, as compared to the non-derivatizedmolecules.

Antibodies

FGF-like polypeptides, fragments, variants, and derivatives may be usedto prepare antibodies using methods known in the art. Thus, antibodiesand antibody fragments that bind FGF-like polypeptides are within thescope of the present invention. Antibodies may be polyclonal,monospecific polyclonal, monoclonal, recombinant, chimeric, humanized,fully human, single chain and/or bispecific.

Polyclonal antibodies directed toward an FGF-like polypeptide generallyare raised in animals (e.g., rabbits or mice) by multiple subcutaneousor intraperitoneal injections of FGF-like polypeptide and an adjuvant.It may be useful to conjugate an FGF-like polypeptide, or a variant,fragment or derivative thereof to a carrier protein that is immunogenicin the species to be immunized, such as keyhole limpet hemocyanin,serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor.Also, aggregating agents such as alum are used to enhance the immuneresponse. After immunization, the animals are bled and the serum isassayed for anti-FGF-like antibody titer.

Monoclonal antibodies directed toward FGF-like polypeptide are producedusing any method that provides for the production of antibody moleculesby continuous cell lines in culture. Examples of suitable methods forpreparing monoclonal antibodies include hybridoma methods of Kohler, etal., Nature 256:495-97 (1975), and the human B-cell hybridoma method,Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications 51-63 (Marcel Dekker 1987).

Also provided by the invention are hybridoma cell lines that producemonoclonal antibodies reactive with FGF-like polypeptides.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(see U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci.U.S.A. 81: 6851-55 (1985).

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source that is non-human.Humanization can be performed following methods known in the art (Jones,et al., Nature 321: 522-25 (1986); Riechmann, et al., Nature 332:323-27(1988); Verhoeyen et al., Science 239:1534-36 (1988)), by substitutingrodent complementarily-determining regions (CDRs) for the correspondingregions of a human antibody.

Also encompassed by the invention are fully human antibodies that bindFGF-like polypeptides, fragments, variants, and/or derivatives. Suchantibodies are produced by immunization with an FGF-like antigen(optionally conjugated to a carrier) of transgenic animals (e.g., mice)that are capable of producing a repertoire of human antibodies in theabsence of endogenous immunoglobulin production. See, e.g., Jakobovits,et al., Proc. Natl. Acad. Sci. U.S.A. 90: 2551-55 (1993); Jakobovits, etal., Nature 362:255-58 (1993); Bruggermann et al., Year in Immuno. 7:33(1993). Human antibodies can also be produced in phage-display libraries(Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks, et al., J. Mol.Biol. 222:581 (1991)).

Chimeric, CDR grafted and humanized antibodies are typically produced byrecombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein above. In a preferred embodiment, the antibodies areproduced in mammalian host cells, such as CHO cells. Fully humanantibodies may be produced by expression of recombinant DNA transfectedinto host cells or by expression in hybridoma cells as described above.

For diagnostic applications, in certain embodiments, anti-FGF-likeantibodies typically will be labeled with a detectable moiety. Thedetectable moiety can be any one that is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the detectablemoiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase or horseradish peroxidase. Bayer, et al.,Meth. Enz. 184: 138-63 (1990).

The anti-FGF-like antibodies of the invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Sola,Monoclonal Antibodies: A Manual of Techniques 147-58 (CRC Press 1987))for detection and quantitation of FGF-like polypeptides. The antibodieswill bind FGF-like polypeptides with an affinity that is appropriate forthe assay method being employed.

Competitive binding assays rely on the ability of a labeled standard(e.g., an FGF-like polypeptide, or an immunologically reactive portionthereof) to compete with the test sample analyte (an FGF-likepolypeptide) for binding with a limited amount of anti FGF-likeantibody. The amount of an FGF-like polypeptide in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected and/or quantitated. In a sandwich assay, the test sampleanalyte is typically bound by a first antibody which is immobilized on asolid support, and thereafter a second antibody binds to the analyte,thus forming an insoluble three part complex. See, e.g., U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan ELISA assay, in which case the detectable moiety is an enzyme.

The anti-FGF-like antibodies of the invention also are useful for invivo imaging, wherein an antibody labeled with a detectable moiety isadministered to an animal, preferably into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.The antibody may be labeled with any moiety that is detectable in ananimal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

The invention also relates to a kit comprising anti-FGF-like antibodiesand other reagents useful for detecting FGF-like polypeptide levels inbiological samples. Such reagents may include a secondary activity, adetectable label, blocking serum, positive and negative control samplesand detection reagents.

Antibodies of the invention may be used as therapeutics. Therapeuticantibodies are generally agonists or antagonists, in that they eitherenhance or reduce, respectively, at least one of the biologicalactivities of an FGF-like polypeptide. In one embodiment, antagonistantibodies of the invention are antibodies or binding fragments thereofwhich are capable of specifically binding to an FGF-like polypeptide,fragment, variant, and/or derivative, and which are capable ofinhibiting or eliminating the functional activity of an FGF-likepolypeptide in vivo or in vitro. In preferred embodiments, an antagonistantibody will inhibit the functional activity of an FGF-like polypeptideat least about 50%, and preferably at least about 80%. In anotherembodiment antagonist antibodies are capable of interacting with anFGF-like binding partner thereby inhibiting or eliminating FGF-likeactivity in vitro or in vivo. Agonist and antagonist anti-FGF-likeantibodies are identified by screening assays described below.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents, rabbits, goats,sheep, or other farm animals, in which the genes encoding nativeFGF-like polypeptide have been disrupted (i.e., “knocked out”) such thatthe level of expression of FGF-like polypeptide is significantlydecreased or completely abolished. Such animals may be prepared usingtechniques and methods such as those described in U.S. Pat. No.5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents, rabbits, goats, sheep, or other farm animals, inwhich a gene encoding a native form of FGF-like polypeptide for thatanimal or a heterologous FGF-like polypeptide gene is overexpressed bythe animal, thereby creating a “transgenic” animal. Such transgenicanimals may be prepared using well known methods such as those describedin U.S. Pat. No. 5,489,743 and PCT Pub. No. WO 94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the FGF-like polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods as described below) to alter the level ofexpression of one or more of the native FGF-like polypeptides.

Such non-human animals may be used for drug candidate screening. Theimpact of a drug candidate on the animal may be measured. For example,drug candidates may decrease or increase expression of the FGF-likepolypeptide gene. In certain embodiments, the amount of FGF-likepolypeptide or an FGF-like polypeptide fragment that is produced may bemeasured after exposure of the animal to the drug candidate. In certainembodiments, one may detect the actual impact of the drug candidate onthe animal. For example, overexpression of a particular gene may resultin, or be associated with, a disease or pathological condition. In suchcases, one may test a drug candidate's ability to decrease expression ofthe gene or its ability to prevent or inhibit a pathological condition.In other examples, production of a particular metabolic product such asa fragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease production of such a metabolic productor its ability to prevent or inhibit a pathological condition.

Modulators of FGF-Like Polypeptide Activity

In some situations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity of FGF-likepolypeptide.

Natural or synthetic molecules that modulate FGF-like polypeptide can beidentified using one or more screening assays, such as those describedbelow. Such molecules may be administered either in an ex vivo manner orin an in vivo manner by local or intravenous injection or by oraldelivery, implantation device, or the like.

The following definition is used herein for describing the assays:

“Test molecule” refers to a molecule that is under evaluation for theability to modulate (i.e., increase or decrease) the activity of anFGF-like polypeptide. Most commonly, a test molecule will interactdirectly with an FGF-like polypeptide. However, it is also contemplatedthat a test molecule may also modulate FGF-like polypeptide activityindirectly, such as by affecting FGF-like gene expression, or by bindingto an FGF-like binding partner (e.g., receptor or ligand). In oneembodiment, a test molecule will bind to an FGF-like polypeptide with anaffinity constant of at least about 10⁻⁶ M, preferably about 10⁻⁸ M,more preferably about 10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds that interact with FGF-likepolypeptides are encompassed by the invention. In certain embodiments,an FGF-like polypeptide is incubated with a test molecule underconditions that permit interaction of the test molecule with an FGF-likepolypeptide, and the extent of the interaction can be measured. The testmolecule may be screened in a substantially purified form or in a crudemixture. Test molecules may be nucleic acid molecules, proteins,peptides, carbohydrates, lipids, or small molecular weight organic orinorganic compounds. Once a set of test molecules has been identified asinteracting with an FGF-like polypeptide, the molecules may be furtherevaluated for their ability to increase or decrease FGF-like polypeptideactivity.

Measurement of the interaction of test molecules with FGF-likepolypeptides may be carried out in several formats, including cell-basedbinding assays, membrane binding assays, solution-phase assays andimmunoassays. In general, test molecules are incubated with an FGF-likepolypeptide for a specified period of time and FGF-like polypeptideactivity is determined by one or more assays described herein formeasuring biological activity.

Interaction of test molecules with FGF-like polypeptides may also beassayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of FGF-like polypeptidescontaining epitope tags as described above may be used in solution andimmunoassays.

In certain embodiments, an FGF-like polypeptide agonist or antagonistmay be a protein, peptide, carbohydrate, lipid, or small molecularweight molecule that interacts with FGF-like polypeptide to regulate itsactivity. Potential protein antagonists of FGF-like polypeptide includeantibodies that interest with active regions of the polypeptide andinhibit or eliminate at least one activity of FGF-like polypeptide.Molecules which regulate FGF-like polypeptide expression may includenucleic acids which are complementary to nucleic acids encoding anFGF-like polypeptide, or are complementary to nucleic acids sequenceswhich direct or control expression of FGF-like polypeptide, and whichact as anti-sense regulators of expression.

In the event that FGF-like polypeptides display biological activitythrough interaction with a binding partner (e.g., a receptor or aligand), a variety of in vitro assays may be used to measure binding ofan FGF-like polypeptide to a corresponding binding partner. These assaysmay be used to screen test molecules for their ability to increase ordecrease the rate and/or the extent of binding of an FGF-likepolypeptide to its binding partner. In one assay, an FGF-likepolypeptide is immobilized by attachment to the bottom of the wells of amicrotiter plate. Radiolabeled FGF-like binding partner (for example,iodinated FGF-like binding partner) and the test molecules can then beadded either one at a time (in either order) or simultaneously to thewells. After incubation, the wells can be washed and counted using ascintillation counter for radioactivity to determine the extent ofbinding to FGF-like protein by its binding partner. Typically, themolecules will be tested over a range of concentrations and a series ofcontrol wells lacking one or more elements of the test assays can beused for accuracy in evaluation of the results. An alternative to thismethod involves reversing the “positions” of the proteins, i.e.,immobilizing FGF-like binding partner to the microtiter plate wells,incubating with the test molecule and radiolabeled FGF-like polypeptide,and determining the extent of FGF-like binding (see, e.g., CurrentProtocols in Molecular Biology, schap. 18 (Ausubel et al., eds., JohnWiley & Sons 1995)).

As an alternative to radiolabeling, an FGF-like polypeptide or itsbinding partner may be conjugated to biotin and the presence ofbiotinylated protein can then be detected using streptavidin linked toan enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase(AP), that can be detected colorometrically, or by fluorescent taggingof streptavidin. An antibody directed to an FGF-like polypeptide or toan FGF-like binding partner and that is conjugated to biotin may also beused and can be detected after incubation with enzyme-linkedstreptavidin linked to AP or HRP

An FGF-like polypeptide and an FGF-like binding partner may also beimmobilized by attachment to agarose beads, acrylic beads, or othertypes of such inert substrates. The substrate-protein complex can beplaced in a solution containing the complementary protein and the testcompound; after incubation, the beads can be precipitated bycentrifugation, and the amount of binding between an FGF-likepolypeptide and its binding partner can be assessed using the methodsdescribed above. Alternatively, the substrate-protein complex can beimmobilized in a column and the test molecule and complementary proteinpassed over the column. Formation of a complex between an FGF-likepolypeptide and its binding partner can then be assessed using any ofthe techniques set forth above, i.e., radiolabeling, antibody binding,or the like.

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases formation of a complex between an FGF-likebinding protein and an FGF-like binding partner is a surface plasmonresonance detector system such as the Biacore assay system (Pharmacia,Piscataway, N.J.). The Biacore system may be carried out using themanufacturer's protocol. This assay essentially involves covalentbinding of either FGF-like polypeptide or an FGF-like binding partner toa dextran-coated sensor chip that is located in a detector. The testcompound and the other complementary protein can then be injected intothe chamber containing the sensor chip either simultaneously orsequentially and the amount of complementary protein that binds can beassessed based on the change in molecular mass which is physicallyassociated with the dextran-coated side of the sensor chip; the changein molecular mass can be measured by the detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease formationof a complex between an FGF-like polypeptide and an FGF-like bindingpartner complex. In these cases, the assays set forth above can bereadily modified by adding such additional test compounds eithersimultaneous with, or subsequent to, the first test compound. Theremaining steps in the assay are as set forth above.

In vitro assays such as those described above may be used advantageouslyto screen rapidly large numbers of compounds for effects on complexformation by FGF-like polypeptide and FGF-like binding partner. Theassays may be automated to screen compounds generated in phage display,synthetic peptide, and chemical synthesis libraries.

Compounds which increase or decrease formation of a complex between anFGF-like polypeptide and an FGF-like binding partner may also bescreened in cell culture using cells and cell lines expressing eitherFGF-like polypeptide or FGF-like binding partner. Cells and cell linesmay be obtained from any mammal, but preferably will be from human orother primate, canine, or rodent sources. The binding of an FGF-likepolypeptide to cells expressing FGF-like binding partner at the surfaceis evaluated in the presence or absence of test molecules and the extentof binding may be determined by, for example, flow cytometry using abiotinylated antibody to an FGF-like binding partner. Cell cultureassays may be used advantageously to further evaluate compounds thatscore positive in protein binding assays described above.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase expression of theFGF-like polypeptide gene. In certain embodiments, the amount ofFGF-like polypeptide or an FGF-like polypeptide fragment that isproduced may be measured after exposure of the cell culture to the drugcandidate. In certain embodiments, one may detect the actual impact ofthe drug candidate on the cell culture. For example, overexpression of aparticular gene may have a particular impact on the cell culture. Insuch cases, one may test a drug candidate's ability to increase ordecrease expression of the gene or its ability to prevent or inhibit aparticular impact on the cell culture. In other examples, production ofa particular metabolic product such as a fragment of a polypeptide, mayresult in, or be associated with, a disease or pathological condition.In such cases, one may test a drug candidate's ability to decreaseproduction of such a metabolic product in a cell culture.

Cell Source Identification Using FGF-like Polypeptide

According to certain embodiments, it may be useful to be able todetermine the source of a certain cell type. For example, it may beuseful to determine the origin of a disease or pathological conditionthat may aid in selecting appropriate therapy. FGF-like polypeptide isspecifically expressed in the liver (and weakly expressed in the lung).In certain embodiments, nucleic acid encoding an FGF-like polypeptidecan be used as a probe to identify liver-derived cells by screening thenucleic acids of the cells with such a probe. In other embodiments, onemay use the FGF-like polypeptide to make antibodies that are specificfor FGF-like polypeptide. Such antibodies can be used to test for thepresence of FGF-like polypeptide in cells, and thus, as a means fordetermining whether such cells were derived from the liver.

FGF-like Polypeptide Compositions and Administration

Therapeutic compositions of FGF-like polypeptides are within the scopeof the present invention. Such compositions may comprise atherapeutically effective amount of an FGF-like polypeptide, fragment,variant, or derivative in admixture with a pharmaceutically acceptableagent such as a pharmaceutically acceptable carrier. The carriermaterial may be water for injection, preferably supplemented with othermaterials common in solutions for administration to mammals. Typically,an FGF-like polypeptide therapeutic compound will be administered in theform of a composition comprising purified polypeptide, fragment,variant, or derivative in conjunction with one or more physiologicallyacceptable agents. Neutral buffered saline or saline mixed with serumalbumin are exemplary appropriate carriers. Preferably, the product isformulated as a lyophilizate using appropriate excipients (e.g.,sucrose). Other standard pharmaceutically acceptable agents such ascarriers, diluents, and excipients may be included as desired. Otherexemplary compositions comprise Tris buffer of about pH 7.0-8.5, oracetate buffer of about pH 4.0-5.5, which may further include sorbitolor a suitable substitute therefor.

FGF-like polypeptide pharmaceutical compositions typically include atherapeutically or prophylactically effective amount of FGF-like proteinin admixture with one or more pharmaceutically and physiologicallyacceptable formulation agents selected for suitability with the mode ofadministration. Suitable formulation materials or pharmaceuticallyacceptable agents include, but are not limited to, antioxidants,preservatives, coloring, flavoring and diluting agents, emulsifyingagents, suspending agents, solvents, fillers, bulking agents, buffers,delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.For example, a suitable vehicle may be water for injection,physiological saline solution, or artificial cerebrospinal fluid,possibly supplemented with other materials common in compositions forparenteral administration. Neutral buffered saline or saline mixed withserum albumin are further exemplary vehicles. The term “pharmaceuticallyacceptable carrier” or “physiologically acceptable carrier” as usedherein refers to a formulation agent(s) suitable for accomplishing orenhancing the delivery of the FGF-like protein as a pharmaceuticalcomposition.

The primary solvent in a composition may be either aqueous ornon-aqueous in nature. In addition, the vehicle may contain otherformulation materials for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, rate of dissolution, orodor of the formulation. Similarly, the composition may containadditional formulation materials for modifying or maintaining the rateof release of FGF-like protein, or for promoting the absorption orpenetration of FGF-like protein.

The FGF-like polypeptide compositions can be administered parentally.Alternatively, the compositions may be administered intravenously orsubcutaneously. When systemically administered, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parentally acceptable aqueous solution. The preparation ofsuch pharmaceutically acceptable protein solutions, with due regard topH, isotonicity, stability and the like, is within the skill of the art.

Therapeutic formulations of FGF-like polypeptide compositions useful forpracticing the present invention may be prepared for storage by mixingthe selected composition having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences (18th Ed., A. R. Gennaro, ed., MackPublishing Company 1990) in the form of a lyophilized cake or an aqueoussolution. Acceptable carriers, excipients or stabilizers preferably arenontoxic to recipients and are preferably inert at the dosages andconcentrations employed, and preferably include buffers such asphosphate, citrate, or other organic acids; antioxidants such asascorbic acid; low molecular weight polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

The optimal pharmaceutical formulation will be determined by one skilledin the art depending upon the intended route of administration, deliveryformat and desired dosage. See, e.g., Remington's PharmaceuticalSciences, 1435-1712 (18th Ed., A. R. Gennaro, ed., Mack PublishingCompany 1990). Such compositions may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent FGF-like protein.

An effective amount of an FGF-like polypeptide composition to beemployed therapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which the FGF-like polypeptide isbeing used, the route of administration, and the condition of thepatient. Accordingly, it may be necessary for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical dosage may range from about 0.1μg/kg to up to about 100 mg/kg or more, depending on the factorsmentioned above. In other embodiments, the dosage may range from 1 μg/kgup to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg; or 0.1 μg/kg upto about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose or as two or more doses (which may or maynot contain the same amount of FGF-like polypeptide) over time, or as acontinuous infusion via implantation device or catheter.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, the type of disorder under treatment, the age and generalhealth of the recipient, will be able to ascertain proper dosing.

The FGF-like polypeptide composition to be used for in vivo parenteraladministration typically must be sterile. This is readily accomplishedby filtration through sterile filtration membranes. Where thecomposition is lyophilized, sterilization using these methods may beconducted either prior to, or following, lyophilization andreconstitution. The composition for parenteral administration ordinarilywill be stored in lyophilized form or in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Effective administration forms, such as (1) slow-release formulations,(2) inhalant mists, or (3) orally active formulations are alsoenvisioned. The FGF-like polypeptide pharmaceutical composition also maybe formulated for parenteral administration. Such parenterallyadministered therapeutic compositions are typically in the form of apyrogen-free, parenterally acceptable aqueous solution comprisingFGF-like polypeptide in a pharmaceutically acceptable vehicle. TheFGF-like polypeptide pharmaceutical compositions also may includeparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc., or the introduction of FGF-like polypeptideinto liposomes. Hyaluronic acid may also be used, and this may have theeffect of promoting sustained duration in the circulation.

A particularly suitable vehicle for parenteral injection is steriledistilled water in which FGF-like polypeptide is formulated as asterile, isotonic solution, properly preserved. Yet another preparationmay involve the formulation of FGF-like polypeptide with an agent, suchas injectable microspheres, bio-erodible particles or beads, orliposomes, that provides for the controlled or sustained release of theprotein product which may then be delivered as a depot injection. Othersuitable means for the introduction of FGF-like polypeptide includeimplantable drug delivery devices that contain the FGF-like polypeptide.

The preparations of the present invention may include other components,for example parenterally acceptable preservatives, tonicity agents,cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, antioxidants and surfactants, as are well known in theart. For example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol and the like. Suitable preservatives include, but are notlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsare for example glycerin, propylene glycol, and polyethylene glycol.Suitable complexing agents are for example caffeine,polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal and the like. The bufferscan be conventional buffers such as borate, citrate, phosphate,bicarbonate, or Tris-HCl.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

A pharmaceutical composition may be formulated for inhalation. Forexample, FGF-like polypeptide may be formulated as a dry powder forinhalation. FGF-like polypeptide inhalation solutions may also beformulated in a liquefied propellant for aerosol delivery. In yetanother formulation, solutions may be nebulized.

It is also contemplated that certain formulations containing FGF-likepolypeptide may be administered orally. FGF-like polypeptide that isadministered in this fashion may be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents may be includedto facilitate absorption of FGF-like polypeptide. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Another preparation may involve an effective quantity of FGF-likepolypeptide in a mixture with non-toxic excipients that are suitable forthe manufacture of tablets. By dissolving the tablets in sterile water,or other appropriate vehicle, solutions can be prepared in unit doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional FGF-like polypeptide formulations will be evident to thoseskilled in the art, including formulations involving FGF-likepolypeptide in combination with one or more other therapeutic agents.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, the Supersaxo et al.description of controlled release porous polymeric microparticles forthe delivery of pharmaceutical compositions (See PCT Pub. No. WO93/15722) the disclosure of which is hereby incorporated by reference.

Regardless of the manner of administration, the specific dose may becalculated according to body weight, body surface area, or organ size.Further refinement of the calculations necessary to determine theappropriate dosage for treatment involving each of the above mentionedformulations is routinely made by those of ordinary skill in the art andis within the ambit of tasks routinely performed by them. Appropriatedosages may be ascertained through use of appropriate dose-responsedata.

The route of administration of the composition is in accord with knownmethods, for example, oral, injection or infusion by intravenous,intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems or implantationdevice which may optionally involve the use of a catheter. Wheredesired, the compositions may be administered continuously by infusion,bolus injection or by implantation device.

One may further administer the present pharmaceutical compositions bypulmonary administration, see, e.g., PCT Pub. WO 94/20069, whichdiscloses pulmonary delivery of chemically modified proteins, hereinincorporated by reference. For pulmonary delivery, the particle sizeshould be suitable for delivery to the distal lung. For example, theparticle size may be from 1 μm to 5 μm. However, larger particles may beused, for example, if each particle is fairly porous.

Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which FGF-like polypeptide has beenabsorbed or encapsulated.

Where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of FGF-like polypeptide maybe directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

FGF-like polypeptide may be administered in a sustained releaseformulation or preparation. Suitable examples of sustained-releasepreparations include semipermeable polymer matrices in the form ofshaped articles, for example, films, or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919, EP Patent No. 58,481), copolymers of L-glutamic acid andgamma ethyl-L-glutamate (Sidman et al, Biopolymers 22: 547-56 (1983)),poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.Res. 15: 167-277 (1981) and Langer, Chem. Tech. 12: 98-105 (1982)),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP Patent No. 133,988).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Epsteinet al., Proc. Natl. Acad. Sci. U.S.A. 82:3688-92 (1985); EP Patent Nos.36,676; 88,046; and 143,949).

The FGF-like polypeptides, fragments thereof, variants, and derivatives,may be employed alone, together, or in combination with otherpharmaceutical compositions. The FGF-like polypeptides, fragments,variants, and derivatives may be used in combination with cytokines,growth factors, antibiotics, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the indication beingtreated.

In some cases, it may be desirable to use FGF-like polypeptidecompositions in an ex vivo manner. Here, cells, tissues, or organs thathave been removed from the patient are exposed to FGF-like polypeptidecompositions after which the cells, tissues and/or organs aresubsequently implanted back into the patient.

In other cases, an FGF-like polypeptide may be delivered throughimplanting into patients certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptides, fragments, variants, or derivatives. Suchcells may be animal or human cells, and may be derived from thepatient's own tissue or from another source, either human or non-human.Optionally, the cells may be immortalized. However, in order to decreasethe chance of an immunological response, it is preferred that the cellsbe encapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow release of the proteinproduct(s) but prevent destruction of the cells by the patient's immunesystem or by other detrimental factors from the surrounding tissues.

Methods used for membrane encapsulation of cells are familiar to theskilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627. A system for encapsulating living cells is described in PCTPub. No. WO 91/10425 (Aebischer et al.). Techniques for formulating avariety of other sustained or controlled delivery means, such asliposome carriers, bio-erodible particles or beads, are also known tothose in the art, and are described. The cells, with or withoutencapsulation, may be implanted into suitable body tissues or organs ofthe patient.

As discussed above, it may be desirable to treat isolated cellpopulations such as stem cells, leukocytes, red blood cells, bonemarrow, chondrocytes, neurons, pancreatic islets, liver cells and thelike with one or more FGF-like polypeptides, variants, derivativesand/or fragments. This can be accomplished by exposing the isolatedcells to the polypeptide, variant, derivative, or fragment directly,where it is in a form that is permeable to or acts upon the cellmembrane.

Additional objects of the present invention relate to methods for boththe in vitro production of therapeutic proteins by means of homologousrecombination and for the production and delivery of therapeuticproteins by gene therapy.

It is further envisioned that FGF-like protein may be produced byhomologous recombination, or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding FGF-like polypeptide. For example, homologous recombinationmethods may be used to modify a cell that contains a normallytranscriptionally silent FGF-like gene, or under expressed gene, andthereby produce a cell that expresses therapeutically efficaciousamounts of FGF-like polypeptide. Homologous recombination is a techniqueoriginally developed for targeting genes to induce or correct mutationsin transcriptionally active genes (Kucherlapati, Prog. in Nucl. AcidRes. and Mol. Biol. 36:301 (1989)). The basic technique was developed asa method for introducing specific mutations into specific regions of themammalian genome (Thomas et al., Cell 44:419-28 (1986); Thomas andCapecchi, Cell 51:503-12, (1987); Doetschman et al., Proc. Natl. Acad.Sci. U.S.A. 85:8583-87 (1988)) or to correct specific mutations withindefective genes (Doetschman et al., Nature 330:576-78 (1987)). Exemplaryhomologous recombination techniques are described in U.S. Pat. No.5,272,071 (EP Patent No. 91 90 3051, EP Publication No. 505 500;PCT/US90/07642, PCT Pub. No. WO 91/09955).

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, and therefore, recombine withother pieces of endogenous DNA through shared homologous regions. Ifthis complementary strand is attached to an oligonucleotide thatcontains a mutation or a different sequence or an additional nucleotide,it too is incorporated into the newly synthesized strand as a result ofthe recombination. As a result of the proofreading function, it ispossible for the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA that mayinteract with the expression of a FGF-like protein. For example, apromoter/enhancer element, a suppresser, or an exogenous transcriptionmodulatory element is inserted in the genome of the intended host cellin proximity and orientation sufficient to influence the transcriptionof DNA encoding the desired FGF-like protein. The control elementcontrols a portion of the DNA present in the host cell genome. Thus, theexpression of FGF-like protein may be achieved not by transfection ofDNA that encodes the FGF-like gene itself, but rather by the use oftargeting DNA (containing regions of homology with the endogenous geneof interest) coupled with DNA regulatory segments that provide theendogenous gene sequence with recognizable signals for transcription ofa FGF-like protein.

In an exemplary method, expression of a desired targeted gene in a cell(i.e., a desired endogenous cellular gene) is altered by theintroduction, by homologous recombination into the cellular genome at apreselected site, of DNA which includes at least a regulatory sequence,an exon and a splice donor site. These components are introduced intothe chromosomal (genomic) DNA in such a manner that this, in effect,results in production of a new transcription unit (in which theregulatory sequence, the exon, and the splice donor site present in theDNA construct are operatively linked to the endogenous gene). As aresult of introduction of these components into the chromosomal DNA, theexpression of the desired endogenous gene is altered.

Altered gene expression, as used herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, increasing expression of a gene which mayinclude expressing a gene that is not expressed at physiologicallysignificant levels in the cell as obtained, changing the pattern ofregulation or induction such that it is different than occurs in thecell as obtained, and reducing (including eliminating) expression of agene which is expressed in the cell as obtained.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) a targeting sequence, (b) aregulatory sequence, (c) an exon, and (d) an unpaired splice-donor site.The targeting sequence in the DNA construct directs the integration ofelements (a)-(d) into a target gene in a cell such that the elements(b)-(d) are operatively linked to sequences of the endogenous targetgene. In another embodiment, the DNA constructs comprise: (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that the elements of (b)-(f) are operatively linked to theendogenous gene. The targeting sequence is homologous to the preselectedsite in the cellular chromosomal DNA with which homologous recombinationis to occur. In the construct, the exon is generally 3′ of theregulatory sequence and the splice-donor site is 3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence of FGF-like polypeptide presented herein, a piece of DNA thatis complementary to a selected region of the gene can be synthesized orotherwise obtained, such as by appropriate restriction of the native DNAat specific recognition sites bounding the region of interest. Thispiece serves as a targeting sequence upon insertion into the cell andwill hybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be backstitched into the newly synthesized daughter strand ofDNA. The present invention, therefore, includes nucleotides encoding aFGF-like molecule, which nucleotides may be used as targeting sequences.

FGF-like polypeptide cell therapy—for example, implantation of cellsproducing FGF-like polypeptide—is also contemplated. This embodimentwould involve implanting into the cells of a patient a construct capableof synthesizing and secreting a biologically active form of FGF-likepolypeptide. Such FGF-like polypeptide-producing cells may be cells thatare natural producers of FGF-like polypeptide or may be recombinantcells whose ability to produce FGF-like polypeptide has been augmentedby transformation with a gene encoding the desired FGF-like molecule orwith a gene augmenting the expression of FGF-like polypeptide. Such amodification may be accomplished by means of a vector suitable fordelivering the gene as well as promoting its expression and secretion.In order to minimize a potential immunological reaction in a patientthat is being administered an FGF-like protein or polypeptide of aforeign species, it is preferred that the natural cells producingFGF-like polypeptide be of human origin and produce human FGF-likepolypeptide. Likewise, it is preferred that the recombinant cellsproducing FGF-like polypeptide are transformed with an expression vectorcontaining a gene encoding a human FGF-like molecule.

Implanted cells may be encapsulated to avoid infiltration of surroundingtissue. Human or non-human animal cells may be implanted in patients inbiocompatible, semipermeable polymeric enclosures or membranes thatallow release of FGF-like polypeptide, but that prevent destruction ofthe cells by the patient's immune system or by other detrimental factorsfrom the surrounding tissue. Alternatively, the patient's own cells,transformed to produce FGF-like polypeptide ex vivo, could be implanteddirectly into the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation ina patient may be accomplished without undue experimentation. Forexample, Baetge et al. (PCT Pub. No. WO 95/05452, the disclosure ofwhich is hereby incorporated by reference) describe membrane capsulescontaining genetically engineered cells for the effective delivery ofbiologically active molecules. The capsules are biocompatible and areeasily retrievable. The capsules encapsulate cells transfected withrecombinant DNA molecules comprising DNA sequences coding forbiologically active molecules operatively linked to promoters that arenot subject to down regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system forencapsulating living cells is described in PCT Pub. No. WO 91/10425(Aebischer et al.). See also, PCT Pub. No. WO 91/10470 (Aebischer etal.); Winn et al., Exper. Neurol. 113:322-29 (1991); Aebischer et al.,Exper. Neurol., 111:269-75 (1991); and Tresco et al., ASAIO 38:17-23(1992).

In vivo and in vitro gene therapy delivery of FGF-like polypeptide isalso envisioned. In vivo gene therapy may be accomplished by introducingthe gene encoding FGF-like polypeptide into cells via local injection ofa polynucleotide molecule or other appropriate delivery vectors (Hefti,J. Neurobiology 25:1418-35 (1994)). For example, a polynucleotidemolecule encoding FGF-like protein may be contained in anadeno-associated virus vector for delivery to the targeted cells (see,e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No. PCT/US95/07178).The recombinant adeno-associated virus (AAV) genome typically containsAAV inverted terminal repeats flanking a DNA sequence encoding FGF-likepolypeptide operably linked to functional promoter and polyadenylationsequences.

Alternative viral vectors include, but are not limited to, retrovirus,adenovirus, herpes simplex virus, and papilloma virus vectors. U.S. Pat.No. 5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631,236 (involvingadenoviral vectors); U.S. Pat. No. 5,672,510 (involving retroviralvectors); and U.S. Pat. No. 5,635,399 (involving retroviral vectorsexpressing cytokines).

Nonviral delivery methods include liposome-mediated transfer, naked DNAdelivery (direct injection), receptor-mediated transfer (ligand-DNAcomplex), electroporation, calcium phosphate precipitation, andmicroparticle bombardment (e.g., gene gun). Gene therapy materials andmethods may also include inducible promoters, tissue-specificenhancer-promoters, DNA sequences designed for site-specificintegration, DNA sequences capable of providing a selective advantageover the parent cell, labels to identify transformed cells, negativeselection systems and expression control systems (safety measures),cell-specific binding agents (for cell targeting), cell-specificinternalization factors, transcription factors to enhance expression bya vector as well as methods of vector manufacture. Such additionalmethods and materials for the practice of gene therapy techniques aredescribed in U.S. Pat. No. 4,970,154 (electroporation techniques); PCTPub. No. WO 96/40958 (nuclear ligands); U.S. Pat. No. 5,679,559(concerning a lipoprotein-containing system for gene delivery); U.S.Pat. No. 5,676,954 (involving liposome carriers); U.S. Pat. No.5,593,875 (concerning methods for calcium phosphate transfection); andU.S. Pat. No. 4,945,050 (wherein biologically active particles arepropelled at cells at a speed whereby the particles penetrate thesurface of the cells and become incorporated into the interior of thecells). Expression control techniques include chemical inducedregulation (see, e.g., PCT Pub. Nos. WO 96/41865 and WO 97/31899), theuse of a progesterone antagonist in a modified steroid hormone receptorsystem (see, e.g., U.S. Pat. No. 5,364,791), ecdysone control systems(see, e.g., PCT Pub. No. WO 96/37609), and positivetetracycline-controllable transactivators (see, e.g., U.S. Pat. No.5,589,362; U.S. Pat. No. 5,650,298; and U.S. Pat. No. 5,654,168).

It is also contemplated that FGF-like polypeptide gene therapy or celltherapy can further include the delivery of a second protein. Forexample, the host cell may be modified to express and release bothFGF-like polypeptide and a second protein, for example insulin-likegrowth factor 1 (IGF-1). Alternatively, both FGF-like polypeptide and asecond protein may be expressed in and released from separate cells.Such cells may be separately introduced into the patient or the cellsmay be contained in a single implantable device, such as theencapsulating membrane described above.

One manner in which gene therapy can be applied is to use the FGF-likegene (either genomic DNA, cDNA, and/or synthetic DNA encoding a FGF-likepolypeptide, or a fragment, variant, or derivative thereof) which may beoperably linked to a constitutive or inducible promoter to form a “genetherapy DNA construct.” The promoter may be homologous or heterologousto the endogenous FGF-like gene, provided that it is active in the cellor tissue type into which the construct will be inserted. Othercomponents of the gene therapy DNA construct may optionally include: DNAmolecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), atissue-specific promoter, enhancers, silencers, DNA molecules capable ofproviding a selective advantage over the parent cell, DNA moleculesuseful as labels to identify transformed cells, negative selectionsystems, cell specific binding agents (as, for example, for celltargeting), cell-specific internalization factors, and transcriptionfactors to enhance expression by a vector as well as factors to enablevector manufacture.

This gene therapy DNA construct can then be introduced into thepatient's cells (either ex vivo or in vivo). One means for introducingthe gene therapy DNA construct is via viral vectors. Suitable viralvectors typically used in gene therapy for delivery of gene therapy DNAconstructs include, without limitation, adenovirus, adeno-associatedvirus, herpes simplex virus, lentivirus, papilloma virus, and retrovirusvectors. Some of these vectors, such as retroviral vectors, will deliverthe gene therapy DNA construct to the chromosomal DNA of the patient'scells, and the gene therapy DNA construct can integrate into thechromosomal DNA; other vectors will function as episomes and the genetherapy DNA construct will remain in the cytoplasm.

Another means to increase endogenous FGF-like polypeptide expression ina cell via gene therapy is to insert one or more enhancer elements intothe FGF-like polypeptide promoter, where the enhancer elements can serveto increase transcriptional activity of the FGF-like polypeptide gene.The enhancer elements used will be selected based on the tissue in whichone desires to activate the gene—enhancer elements known to conferpromoter activation in that tissue will be selected. For example, if agene encoding an FGF-like polypeptide is to be “turned on” in T-cells,the lck promoter enhancer element may be used. Here, the functionalportion of the transcriptional element to be added may be inserted intoa fragment of DNA containing the FGF-like polypeptide promoter (andoptionally, inserted into a vector and/or 5′ and/or 3′ flankingsequences, etc.) using standard cloning techniques. This construct,known as a “homologous recombination construct,” can then be introducedinto the desired cells either ex vivo or in vivo.

Gene therapy can be used to decrease FGF-like polypeptide expression bymodifying the nucleotide sequence of the endogenous promoter. Suchmodification is typically accomplished via homologous recombinationmethods. For example, a DNA molecule containing all or a portion of thepromoter of the FGF-like gene selected for inactivation can beengineered to remove and/or replace pieces of the promoter that regulatetranscription. Here, for example, the TATA box and/or the binding siteof a transcriptional activator of the promoter may be deleted usingstandard molecular biology techniques; such deletion can inhibitpromoter activity thereby repressing transcription of the correspondingFGF-like gene. Deletion of the TATA box or transcription activatorbinding site in the promoter may be accomplished by generating a DNAconstruct comprising all or the relevant portion of the FGF-likepolypeptide promoter (from the same or a related species as the FGF-likegene to be regulated) in which one or more of the TATA box and/ortranscriptional activator binding site nucleotides are mutated viasubstitution, deletion and/or insertion of one or more nucleotides suchthat the TATA box and/or activator binding site has decreased activityor is rendered completely inactive. This construct, which also willtypically contain at least about 500 bases of DNA that correspond to thenative (endogenous) 5′ and 3′ DNA sequences adjacent to the promotersegment that has been modified, may be introduced into the appropriatecells (either ex vivo or in vivo) either directly or via a viral vectoras described above. Typically, integration of the construct into thegenomic DNA of the cells will be via homologous recombination, where the5′ and 3′ DNA sequences in the promoter construct can serve to helpintegrate the modified promoter region via hybridization to theendogenous chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit the activity of one or more FGF-like polypeptides. For example,antisense DNA or RNA molecules, which have a sequence that iscomplementary to at least a portion of the selected FGF-like polypeptidegene can be introduced into the cell. Typically, each such antisensemolecule will be complementary to the start site (5′ end) of eachselected FGF-like gene. When the antisense molecule then hybridizes tothe corresponding FGF-like mRNA, translation of this mRNA is prevented.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more FGF-like polypeptides. Inthis situation, the DNA encoding a mutant full length or truncatedpolypeptide of each selected FGF-like polypeptide can be prepared andintroduced into the cells of a patient using either viral or non-viralmethods as described above. Each such mutant is typically designed tocompete with endogenous polypeptide in its biological role.

Uses of FGF-Like Nucleic Acids and Polypeptides

Nucleic acid molecules of the invention may be used to map the locationsof the FGF-like gene and related genes on chromosomes. Mapping may bedone by techniques known in the art, such as PCR amplification and insitu hybridization.

The nucleic acid molecules are also used as anti-sense inhibitors ofFGF-like polypeptide expression. Such inhibition may be effected bynucleic acid molecules that are complementary to and hybridize toexpression control sequences (triple helix formation) or to FGF-likemRNA. Anti-sense probes may be designed by available techniques usingthe sequence of the FGF-like genes disclosed herein. Anti-senseinhibitors provide information relating to the decrease or absence of anFGF-like polypeptide in a cell or organism.

Hybridization probes may be prepared using an FGF-like gene sequence asprovided herein to screen cDNA, genomic or synthetic DNA libraries forrelated sequences. Regions of the DNA and/or amino acid sequence ofFGF-like polypeptide that exhibit significant identity to knownsequences are readily determined using sequence alignment algorithmsdisclosed above and those regions may be used to design probes forscreening.

The nucleic acid molecules of the invention may be used for genetherapy. Nucleic acid molecules that express FGF-like polypeptide invivo provide information relating to the effects of the polypeptide incells or organisms.

FGF-like nucleic acid molecules, fragments, variants, and/or derivativesthat do not themselves encode biologically active polypeptides may beuseful as hybridization probes in diagnostic assays to test, eitherqualitatively or quantitatively, for the presence of FGF-like DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

FGF-like polypeptides, fragments, variants, and/or derivatives may beused to prevent or treat cirrhosis or other toxic insult of the liver;inflammatory bowel disease, mucositis, Crohn's disease, or othergastrointestinal abnormality; diabetes; obesity; neurodegenerativediseases; wounds; damage to the corneal epithelium, lens, or retinaltissue; damage to renal tubules as a result of acute tubular necrosis;hematopoietic cell reconstitution following chemotherapy; wastingsyndromes (for example, cancer associated cachexia), multiple sclerosis,myopathies; short stature, delayed maturation, excessive growth (forexample, acromegaly), premature maturation; alopecia; diseases orabnormalities of androgen target organs; infantile respiratory distresssyndrome, bronchopulmonary dysplasia, acute respiratory distresssyndrome, or other lung abnormalities; tumors of the eye or othertissues; atherosclerosis; hypercholesterolemia; diabetes; obesity;stroke; osteoporosis; osteoarthritis; degenerative joint disease; muscleatrophy; sarcopenia; decreased lean body mass; baldness; wrinkles;increased fatigue; decreased stamina; decreased cardiac function; immunesystem dysfunction; cancer; Parkinson's disease; senile dementia;Alzheimer's disease; and decreased cognitive function.

FGF-like polypeptide fragments, variants, and/or derivatives, whetherbiologically active or not, are useful for preparing antibodies thatbind to an FGF-like polypeptide. The antibodies may be used for in vivoand in vitro diagnostic purposes, including, but not limited to, use inlabeled form to detect the presence of FGF-like polypeptide in a bodyfluid or cell sample. The antibodies may also be used to prevent ortreat conditions that may be associated with an increase in FGF-likepolypeptide expression or activity. The antibodies may bind to anFGF-like polypeptide so as to diminish or block at least one activitycharacteristic of an FGF-like polypeptide, or may bind to a polypeptideto increase an activity.

A deposit of cDNA encoding FGF-like polypeptide has been made with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 on Sep. 3, 1999 and having accession No. PTA-626.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

Example 1 Cloning of the Murine FGF-like Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding rat FGF-likepolypeptide.

Sequences encoding the murine FGF-like polypeptide were isolated from amouse regenerating liver cDNA library by screening the library in a kFGFsignal trap system (U.S. patent application Ser. No. 09/026,958). Thisprimary screening technique enriched for clones encoding signalpeptide-containing secreted proteins.

A primary library (Tmrl1) was constructed in the kFGF vector as follows.Regenerating mouse liver was removed 24 hours after partial hepatectomy,and poly A+ RNA prepared using a commercially available RNA extractionkit and mRNA purification kit (Pharmacia Biotech). A cDNA library wasprepared using the SuperScript™ Plasmid System for cDNA Synthesis andPlasmid Cloning (Gibco BRL) with some modifications. First strandreactions were performed using 3 μg of poly A+ RNA and 500 μg of theprimer5′-G-G-A-A-G-G-A-A-A-A-A-A-G-C-G-G-C-C-G-C-A-A-C-A-N-N-N-N-N-N-N-N-N-3′(SEQ ID NO: 34). Following second strand synthesis, the cDNA was ligatedto a Sal I adapter, digested with Not I, and then size fractionated byagarose gel electrophoresis. Fractionated cDNA, ranging from 0.2 to 0.8kb in size, was purified using a Qiagen gel extraction kit and thenligated to the kFGF vector as follows. In a 20 μl reaction, 50 μg ofvector DNA (previously digested with Sal I and Not I) was mixed with 20μg of the purified cDNA, 1× ligase buffer, and 1 ml of T4 DNA ligase at16° C. for 20 hours.

The product of this ligation was precipitated and introduced into E.coli by electroporation, after which transformed bacterial cells weregrown in 5 ml SOC medium at 37° C. for 1 hour and then frozen at −80° C.in 10% glycerol. This constituted the primary Tmrl1 library. Plasmid DNAfrom the primary Tmrl1 cDNA library was prepared from pools of 50,000colonies grown on LB/agar plates using standard procedures. Ten poolswere prepared and plasmid DNA was isolated from the pools using Qiagenmaxi prep kits.

The plasmid DNA that was recovered was subsequently introduced into NIH3T3 cells by calcium phosphate transfection. In each reaction 100 ng ofplasmid DNA from each pool was used to transfect approximately 2×10⁵cells in one 35 mm plate. After 24 hours, the cells were then split intofive 100 mm plates, grown in normal medium for one day, and then grownin low serum medium for 13 days. Approximately 4000 total colonies wereobtained following growth in the low serum media.

Signal peptide-enriched regenerating cDNA molecules were recovered fromthe transfected cells as follows. Cells were released from the plates bythe addition of 2 ml trypsin-EDTA and incubation at 37° C. for 5minutes, followed by gentle swirling. Released cells were transferred to50 ml conical tubes with 2 ml of fetal calf serum and centrifuged at1000 rpm for 5 minutes to pellet the cells. Cell pellets of no more than1 gram were lysed with 20 ml of TRIZOL reagent (BRL), homogenized for 30seconds, and then extracted with 4 ml of chloroform. The tubes werecentrifuged at 4000 rpm for 30 minutes and the aqueous phase wastransferred to a new tube. RNA was precipitated by adding 10 mlisopropanol, mixing, and then centrifuging for 30 minutes at 4200 rpm.The RNA pellet was washed with 10 ml of 70% ethanol, briefly dried, andthen the pellet was resuspended in 0.5 ml TE buffer. Poly A+ RNA wasprepared by using a commercially available mRNA purification kit(Pharmacia). After eluting poly A+ RNA from the column in 750 μl of TEbuffer, the sample was then ethanol precipitated in two 1.5 mlmicrocentrifuge tubes by adding 40 μl of sample buffer and 1 ml ofethanol and incubating overnight at −70° C.

The cDNA inserts of positive clones were rescued by RT-PCR as follows.First strand synthesis was performed using the SuperScript™preamplification system (BRL). A mixture containing 15 μg of poly A+ RNAfrom selected 3T3 colonies and 15 μl (2 μM) of vector primer(5′-A-A-T-C-C-G-A-T-G-C-C-C-A-C-G-T-T-G-C-A-G-T-A-3′; SEQ ID NO: 35) wasprepared and then incubated at 70° C. for 10 minutes followed byequilibration at 50° C. A premixture containing 2.5 μl 10× buffer, 2.5μl 25 mM MgCl₂, 1.3 μl 10 mM dNTPs, and 2.5 μl 0.1 M dithiothreitol wasthen added to the poly A+ RNA/primer mixture, after which 1.2 μl ofreverse transcriptase was added and the reaction incubated at 50° C. for1 hour. The reaction was stopped by heating at 70° C. for 15 minutes.

Following first strand synthesis, RNA was digested with 1 μl RNase H at37° C. for 20 minutes. PCR was performed using Pfu polymerase (PerkinElmer) as follows. In a total volume of 100 μl, 2 μl of the first strandreaction was added to 1×Pfu buffer, 0.5 μM each of the amplificationprimers (5′-A-A-A-A-T-C-T-T-A-G-A-C-C-G-A-C-G-A-C-T-G-T-G-T-T-T-3′; SEQID NO: 36; and 5′-G-A-G-T-C-T-C-C-G-C-A-G-C-C-T-T-T-T-G-A-G-G-3′; SEQ IDNO: 37), 0.2 mM dNTPs, 5% DMS, and 2.5 units of Pfu polymerase. Theamplification reaction was performed at 95° C. for 1 minute for 1 cycle;95° C. for 30 seconds, 66° C. for 45 seconds, and 72° C. for 30 cycles;and 72° C. for 10 minutes for 1 cycle. PCR products were purified byphenol/chloroform extraction followed by ethanol precipitation, and thenwere digested with Not I and Sal I. Small digestion products and PCRprimers were removed from the reaction using SizeSep 400 Spun columns.

Clones identified in the primary screen were subsequently analyzed in asecondary secretion assay. This secondary screening technique utilized avector containing a truncated placental alkaline phosphatase (PLAP), inwhich the native signal peptide had been removed, as a secretionreporter gene. Heterologous cDNA fragments were tested for the presenceof signal peptide secretion sequences by inserting individual sequencesimmediately upstream of the truncated PLAP gene and then transfectingCOST cells with the test constructs. Inserted cDNA sequences encoding asignal peptide, when inserted in frame with the PLAP reporter sequence,lead to the formation of a fusion protein that can be secreted from thetransfected cells.

The PLAP reporter construct was generated as follows. Human placenta RNAwas subjected to RT/PCR amplification under standard conditions togenerate a DNA fragment encoding a truncated human PLAP protein. The RNAwas transcribed by reverse transcriptase using oligo d(T) as primer, andthen PCR amplified with PLAP specific primers to produce a DNA productwhich encodes a PLAP sequence corresponding to amino acids 22 to 536(Millan, J. Biol. Chem. 261(7):3112-15 (1986)). The PLAP amplificationprimers(5′-A-C-T-G-G-C-G-G-C-C-G-C-A-G-G-C-A-T-C-A-T-C-C-C-A-G-T-T-G-A-G-G-A-G-3′;SEQ ID NO: 38; and5′-A-C-T-G-G-T-C-A-C-T-C-G-A-G-G-G-T-A-C-C-T-T-A-G-C-T-A-G-C-C-C-C-C-G-G-G-3′;SEQ ID NO: 39) were designed to contain Not I and Kpn I restrictionsites, respectively, in order to facilitate the ligation of the PLAPfragment into the pcDNA3.1 vector (generating the vector pcDNA3.1/PLAP).

The Tmrl1 secondary library was constructed in pcDNA3.1/PLAP as follows.The cDNA inserts from clones in the primary Tmrl1 library were recoveredfrom 3T3 cell colonies using the PCR amplification primers andconditions noted above. Recovered PCR products were then ligated intothe pcDNA3.1/PLAP vector which had been digested with the Xho I and NotI restriction enzymes. Ligation products were then transformed into theE. Coli strain DH10B to generate the secondary Tmrl1 library.

To assay heterologous cDNA fragments for the presence of signal peptidesecretion sequences individual colonies were first selected from a lowdensity plating of the secondary Tmrl1 library on agar plates and placedinto the wells of a standard 96-well plate containing bacterial growthmedium. The cultures were then grown to saturation and plasmid DNA wasprepared from each culture by standard procedures. Plasmid DNA preparedas such was used to transfect COS7 cells as described below.

COS7 cells were seeded in 96-well flat-bottom plates at a concentrationof 6000 cells per well in 200 μl of a growth medium consisting ofDMEM-HG, 10% fetal bovine serum and 1×PSG (penicillin, streptomycin andgentamycin). Following seeding with COS7 cells, the plates wereincubated for 24 hours at 37° C. in a CO₂ incubator. The cells in eachwell were subsequently transfected with 500 ng of plasmid DNA recoveredfrom selected secondary Tmrl1 library clones using Superfect reagent(Qiagen). Transfection reactions were allowed to proceed for two hours,after which the transfected cells were washed with 200 of phenolred-free, serum-free DMEM and then incubated with 100 μl of phenolred-free, serum-free DMEM and glutamate for 24 hours at 37° C. in a CO₂incubator. Following incubation, 100 μl of a solution containing 200 μM4-methylumbelli-feryl phosphate (Molecular Probes) in 1M diethanolamine,10 mM homo-arginine, 1 mM MgCl₂, and 1 mg/ml BSA was added to each well,and incubation continued for 1 hour at 37° C. The product of thealkaline phosphatase reaction was then read in a fluorometer at 360/460nm.

A single clone (tmrl1-00001-e9), yielding an increased fluorescencereadout in the PLAP assay and possessing a computer-predicted signalpeptide sequence and homology to the FGF family, was identified in thesecondary Trull library screen. This clone was subsequently used as aprobe to isolate a full-length cDNA for murine FGF-like polypeptide froma mouse liver cDNA library. A mouse liver full-length cDNA library wasconstructed using standard techniques. Essentially, oligo d(T) was usedto prime first strand synthesis from mRNA isolated from regeneratingmouse liver and full-length cDNA sequences were then cloned into thepSPORT (Gibco BRL) vector using the Superscript Plasmid System for cDNASynthesis and Plasmid Cloning (Gibco BRL).

In a primary screen of the mouse liver cDNA library, 50,000 colonieswere plated on LB/Ampicillin plates and then transferred tonitrocellulose filters. The filters were screened with a mixture ofprobes derived from clones identified in the kFGF signal trap andsecretion assay screenings. This pool of probes included a 339 bp NotI-Xba I fragment isolated from the tmrl1-00001-e9 clone. The filterswere first prehybridized for 2 hours at 42° C. in a hybridizationsolution consisting of 50% formamide, 5×Denhardt's, 5×SSC, 0.5% SDS, and100 μg/ml salmon sperm DNA. Following prehybridization, the filters wereincubated with the ³²P-dCTP labeled probes overnight at 42° C. in freshhybridization solution. The filters were then washed with 0.1×SSC/0.1%SDS at 65° C. and were subsequently analyzed by autoradiographyovernight at −80° C. From this initial screen, 92 positive clones wereidentified.

The positive clones isolated in the primary screen were pooled andrescreened with the 339 bp tmrl1-00001-e9 fragment alone. In thissecondary screening, 6000 colonies derived from the clone pool wereplated on LB/Ampicillin plates and then transferred to nitrocellulosefilters. The hybridization conditions used in the secondary screen werethe same as those used for the primary screen. Three individual clones(1E, 1E-4, and 1E-6) were identified in the secondary screen.

Restriction digestion of the three individual clones identified in thesecondary screen indicated that each contained a 1.6 kb insert. Primerscorresponding to the 5′(5′-T-G-G-A-A-T-G-G-A-T-G-A-G-A-T-C-T-A-G-A-G-3′; SEQ ID NO: 7) and 3′(5′-C-T-A-G-A-T-T-C-A-G-G-A-A-G-A-G-T-C-A-3′; SEQ ID NO: 8) ends of thecoding sequence encoded by this insert were designed following partialsequence analysis of clone 1E. These primers were used in a polymerasechain reaction (PCR) amplification of clone 1E plasmid DNA. Theamplification reaction was performed at 94° C. for 1 minute for 1 cycle;94° C. for 15 seconds and 65° C. for 1.5 minutes for 35 cycles; and 72°C. for 10 minutes for 1 cycle. A PCR product of approximately 650 bp wasobtained following amplification.

Following purification from a 1% agarose gel using a Qiagen gelextraction kit, the PCR product was sequenced. Sequence analysis of thisamplification product indicated that the cDNA clones from which the PCRprimers were derived contained a gene comprising a 630 bp open readingframe encoding a protein of 210 amino acids. FIG. 1 illustrates thenucleotide sequence of the murine FGF-like gene (SEQ ID NO: 3) and thededuced amino acid sequence of murine FGF-like protein (SEQ ID NO: 4).Subsequent sequence analysis of clone 1E also established that the openreading frame of the cDNA clones identified in the secondary screeningwas interrupted by two intron sequences. Computer analysis using theFASTA program of the Swissprot database indicated that this open readingframe encoded a polypeptide that is most closely related (39% identical)to FGF-6 (FIGS. 3A-3D).

Computer analysis, using the SIGNALP program (Center for BiologicalSequence Analysis, The Technical University of Denmark), also indicatedthat the murine FGF-like polypeptide possessed a potential signalpeptide at its amino terminus(M-E-W-M-R-S-R-V-G-T-L-G-L-W-V-R-L-L-L-A-V-F-L-L-G-V-Y-Q-A; SEQ ID NO:40; as underlined in FIG. 1). The initial translation product of murineFGF-like polypeptide has a calculated molecular weight of 23,237, notincluding possible post-translational modifications. After removal ofthe predicted 29 amino acid signal peptide sequence, the remainingpredicted mature protein of 181 amino acids has a calculated molecularweight of 19,876. No predicted N-linked glycosylation sites wereidentified in the protein and the protein does not possess any dibasicprotease processing sites.

Example 2 Cloning of the Human FGF-like Polypeptide Gene

Generally, materials and methods as described in Sambrook et al. suprawere used to clone and analyze the gene encoding human FGF-likepolypeptide.

Sequences encoding the human FGF-like polypeptide were isolated byscreening a human cDNA library with a probe derived from the murineFGF-like polypeptide gene. A 460 bp probe was generated by PCRamplification of murine FGF-like polypeptide cDNA using the followingprimers: 5′-T-G-G-A-A-T-G-G-A-T-G-A-G-A-T-C-T-A-G-A-G-3′; SEQ ID NO: 7)and 3′(5′-C-A-T-T-G-C-G-G-C-C-G-C-T-C-A-A-G-A-T-G-C-A-A-A-A-C-G-C-A-G-T-G-3′;SEQ ID NO: 9) in reactions containing ³²P-dCTP. The amplificationreaction was performed at 94° C. for 1 minute for 1 cycle; 94° C. for 15seconds and 65° C. for 1.5 minutes for 35 cycles; and 72° C. for 10minutes for 1 cycle.

The 460 bp murine probe was used to isolate a full-length cDNA for humanFGF-like polypeptide from a human liver cDNA library. A human liverfull-length cDNA library was constructed using standard techniques.Essentially, oligo d(T) was used to prime first strand synthesis frommRNA obtained from Clontech (Palo Alto, Calif.) and full-length cDNAsequences were then cloned into the pSPORT (Gibco BRL) vector using theSuperscript Plasmid System for cDNA Synthesis and Plasmid Cloning (GibcoBRL).

In a primary screen of the human liver cDNA library, 550,000 colonieswere plated on LB/Ampicillin plates and then transferred tonitrocellulose filters. The filters were first prehybridized for 30minutes at 60° C. in ExpressHyb solution (Clontech), and then wereincubated with the ³²P-dCTP labeled murine FGF-like cDNA probe overnightat 60° C. in fresh ExpressHyb solution. Following hybridization, thefilters were washed twice for 30 minutes at room temperature in2×SSC/0.1% SDS, twice for 30 minutes at 60° C. in 1×SSC/0.1% SDS, twicefor 30 minutes at 65° C. in 0.1×SSC/0.1% SDS, and then the filters wereanalyzed by autoradiography overnight at −80° C. Positive clonesidentified in the primary screen were then rescreened, and fourindependent clones were recovered following the secondary screen.Plasmid DNA for these four clones was prepared and sequenced.

Sequence analysis indicated that the four clones contained inserts ofeither 1.2 kb or 1.8 kb. The 1.2 kb cDNA insert contained a genecomprising a 627 bp open reading frame encoding a protein of 209 aminoacids. FIGS. 2A-2B illustrate the nucleotide sequence of the humanFGF-like gene (SEQ ID NO: 1) and the deduced amino acid sequence ofhuman FGF-like protein (SEQ ID NO: 2). While the 1.8 kb cDNA insertcontained the same open reading frame as encoded by the 1.2 kb insert,the open reading frame of this insert was additionally interrupted by anintron corresponding in location to the second intron found in some ofthe murine cDNA clones isolated in Example 1.

FIGS. 3A-3D illustrate the amino acid sequence alignment of human

FGF-like protein, murine FGF-like protein, and other members of the FGFfamily. The human FGF-like polypeptide is 76% identical to the murineFGF-like protein. Computer analysis, using the SIGNALP program (Centerfor Biological Sequence Analysis, The Technical University of Denmark),indicated that the human FGF-like polypeptide also possessed a potentialsignal peptide at its amino terminus(M-D-S-D-E-T-G-F-E-H-S-G-L-W-V-S-V-L-A-G-L-L-L-G-A-C-Q-A; SEQ ID NO: 41;as underlined in FIGS. 2A-2B). The initial translation product of humanFGF-like polypeptide has a calculated molecular weight of 22,284, notincluding possible post-translational modifications. After removal ofthe predicted 28 amino acid signal peptide sequence, the remainingpredicted mature protein of 181 amino acids has a calculated molecularweight of 19,395. No predicted N-linked glycosylation sites wereidentified in the protein and the protein does not possess any dibasicprotease processing sites.

Example 3 FGF-like mRNA Expression

Expression of murine FGF-like mRNA was examined on a murine multipletissue Northern blot (Clontech) using a ³²P-dCTP labeled murine FGF-likecDNA probe. The probe consisted of a 391 bp fragment isolated from thetmrl1-00001-e9 clone by restriction digestion with Xba I and Not I. Theblot was first prehybridized for 2 hours at 42° C. in a hybridizationsolution consisting of 50% formamide, 5×Denhardt's, 6×SSC, 0.5% SDS, and100 μg/ml salmon sperm DNA. Following prehybridization, the filters wereincubated with the ³²P-dCTP labeled murine FGF-like cDNA probe overnightat 42° C. in fresh hybridization solution. The filters were then washedwith 0.1×SSC/0.1% SDS at 65° C. and were subsequently analyzed byautoradiography overnight at −80° C. Two transcripts, of approximately1.35 kb and 1.8 kb, were detected in murine liver (FIG. 4A). The 1.35 kbtranscript showed the predominant expression.

Expression of human FGF-like mRNA was examined on a Human RNA MasterBlot™ (Clontech) and a human multiple tissue Northern blot (Clontech)using a ³²P-dCTP labeled human FGF-like cDNA probe. The probe consistedof a 660 bp PCR product derived from the human FGF-like protein codingregion using the primers:5′-C-T-A-C-TA-A-A-G-C-T-T-C-C-A-C-C-A-T-G-G-A-C-T-C-G-G-A-C-G-A-G-A-C-C-G-3′;SEQ ID NO: 12; and5′A-T-T-C-A-T-G-C-G-G-C-C-G-C-G-G-A-A-G-C-G-T-A-G-C-T-G-G-G-G-C-T-T-C-3′;SEQ ID NO: 13, and the human cDNA clone described above as a template.The amplification reaction was performed at 94° C. for 1 minute for 1cycle; 94° C. for 15 seconds, 60° C. for 15 seconds, and 72° C. for 1minute for 35 cycles; and 72° C. for 10 minutes for 1 cycle. The blotswere first prehybridized for 1 hour at 65° C. in ExpressHyb solution(Clontech), and then were incubated with the ³²P-dCTP labeled humanFGF-like cDNA probe overnight at 65° C. in fresh ExpressHyb solution.Following hybridization, the filters were washed twice for 30 minutes atroom temperature in 2×SSC/0.1% SDS, twice for 30 minutes at 65° C. in0.1×SSC/0.1% SDS, and then the filters were analyzed by autoradiographyovernight at −80° C. Two transcripts, of approximately 1.2 kb and 2 kb,were detected in human liver on the multiple tissue Northern blot 9(FIG. 4B). Strong expression in adult liver and weak expression in lungand fetal liver was detected on the Human RNA Master Blot™ (FIG. 4C).

Example 4 FGF-like mRNA In Situ Analysis

In situ hybridization was performed with a 648 bp antisense RNA probespanning the coding region of murine FGF-like polypeptide. The probe wastranscribed from a linearized pCR2.1 TOPO plasmid containing theFGF-like cDNA insert using T7 RNA polymerase and ³³P-UTP.

A panel of normal embryonic and adult mouse tissues were fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Prior toin situ hybridization, tissues were permeabilized with 0.2M HCl,followed by digestion with Proteinase K, and acetylation withtriethanolamine and acetic anhydride. Sections were hybridized with the³³P-labeled riboprobe overnight at 55° C., then subjected to a highstringency wash in 0.1×SSC at 60° C. Slides were dipped in Kodak NTB2emulsion, exposed at 4° C. for 2-3 weeks, developed, and counterstainedwith hematoxylin and eosin. Sections were examined with darkfield andstandard illumination to allow simultaneous evaluation of tissuemorphology and hybridization signal.

The strongest overall expression was noted in the pancreas with a strongsignal being detected over the islets and a lower, more diffuse signalover the acinar portion of the pancreas. The liver displayed a moderatelevel of diffuse signal, indicative of moderate hepatocellularexpression of FGF-like polypeptide. Significant signal was also presentover spermatogonia within the seminiferous tubules in the testis andover cells in the thymic medulla. A low level of diffuse signal wasdetected in kidney, spleen, pituitary, and white and brown adiposetissue.

Example 5 Production of FGF-like Polypeptides in Mammalian Cells

Both the human and murine FGF-like polypeptides were expressed as fusionproteins with a human immunoglobulin IgG heavy chain Fc region at theircarboxyl terminus. Template DNA sequences encoding human or murineFGF-like polypeptide were amplified by PCR using primers correspondingto the 5′ and 3′ ends of the sequence (Table II). The resulting PCRproducts corresponded to the coding region of either the human or murineFGF-like polypeptide, minus the translation termination codon. Inaddition, the primers were designed to incorporate a Hind IIIrestriction endonuclease site at the 5′ end of the PCR product and a NotI site at the 3′ end of the product.

TABLE II Primers Used in Recombinant Protein Expression Murine 5′5′-CTACTAAAGCTTCCACCATGGAATGGATGAGATCTA G-3′ (SEQ ID NO: 10) Murine 3′5′ATTCATGCGGCCGCGGACGCATAGCTGGGGCTT-3′ (SEQ ID NO: 11) Human 5′5′-CTACTAAAGCTTCCACCATGGACTCGGACGAGACC G-3′ (SEQ ID NO: 12) Human 3′5′-ATTCATGCGGCCGCGGAAGCGTAGCTGGGGCTTC-3′ (SEQ ID NO: 13)

The human and murine FGF-like protein-Fc fusion constructs weregenerated by first cleaving the PCR products with Hind III and Not I andthen ligating the fragments in frame to a DNA fragment encoding the HFcchain of human immunoglobulin IgG. The FGF-like protein-Fc insert wasthen ligated into the pCEP4 mammalian expression vector (Invitrogen).These ligations were transformed into the E. coli strain DH10 byelectroporation and transformants selected for ampicillin resistance.Following sequence analysis of selected transformants, large-scaleplasmid stocks were prepared for tissue culture transfection. PlasmidDNA for selected ampicillin resistant colonies was prepared andsequenced to confirm that the clone contained the desired insert.

To conduct functional studies on FGF-like protein, human or murineFGF-like protein-Fc fusion expression constructs were introduced into293-EBNA (Invitrogen) cells using SuperFect™ transfection reagent(Qiagen). The conditioned medium was harvested 48 hours after transienttransfection. Western blot analysis, using an anti-human Fc antibody,confirmed that the conditioned media contained human or murineFGF-like/Fc fusion polypeptides.

Conditioned medium was purified by affinity chromatography as describedbelow. The medium was first passed through a 0.2 μm filter. Protein Acolumns (Pharmacia) were equilibrated with ImmunoPure Gentle BindingBuffer (Pierce, Rockford, Ill.), and then loaded with the filteredmedium. The column was washed with ImmunoPure Gentle Binding Bufferuntil the absorbance at 280 nm reached a baseline. FGF-like/Fc proteinwas eluted from the column with ImmunoPure Gentle Binding Buffer.Fractions containing FGF-like/Fc protein were pooled, dialyzed inTris-buffered saline (TBS) followed by Phosphate-buffered saline (PBS),and stored at 4° C. Gel electrophoretic analysis confirmed that thepooled fractions contained a purified protein of the expected molecularweight of about 60 kD.

Example 6 Production and Analysis of Transgenic Mice OverexpressingFGF-like Polypeptide

Transgenic mice overexpressing mouse FGF-like polypeptide (SEQ ID NO: 4)from the human apolipoprotein E promoter were generated as previouslydescribed (see Simonet et al., 1997, Cell 89:309-19). Seven mice (fourmales and three females) which were transgenic for the murine FGF-likegene (SEQ ID NO: 3) and five non-transgenic littermates (two males andthree females) underwent necropsy and pathological analysis at 6-8 weeksof age. All of the mice were injected with 50 mg/kg of BrdU one hourprior to harvest, then were radiographed and sacrificed. Body andselected organ weights were measured, blood was drawn for hematology andserum chemistries, and organs were harvested for histological analysisand BrdU labeling.

All of the transgenic mice were under 20 gm body weight while all of thenon-transgenic mice were over 20 gm body weight (p<0.0001). In addition,transgenic mice had statistically significantly lower liver (p=0.0011)and spleen weights (p=0.0039) and a higher thymic (p=0.0118) weight (asa percent of body weight) than their non-transgenic counterparts. Forthe transgenic mice, body weight was 67% of wild type. Liver, spleen,and thymus weights, as a percentage of body weight, were, respectively,85%, 63% and 170% of wild type. All female transgenic mice also hadsmall uteri and oviducts and ovaries that lacked corpora lutea andexhibited little follicular development. In summary, transgenic mice hada phenotype that is best characterized as stunted growth with smalllivers, spleens, and poorly developed ovaries, and enlarged (probablynot involuted) thymuses. These changes are most consistent withoverexpression of FGF-like polypeptide in the transgenics causinginhibited or delayed maturation in comparison with their age-matchednon-transgenic littermates.

Example 7 Analysis of One-Year-Old FGF-like Polypeptide Transgenic Mice

Necropsy and pathological analysis was performed on one-year-oldFGF-like polypeptide transgenics (five males and five females) andnon-transgenic (three males and two females) littermates that had beenproduced in the experiments described in Example 6. FGF-like polypeptidetransgenics continued to demonstrate an abnormal phenotype generallycharacterized as inhibited or delayed maturation. These observed effectsincluded reduced body weight (48% of wild type) and, in the females,poorly developed ovaries with lack of significant folliculardevelopment. Liver, spleen, and thymus weights as a percentage of bodyweight had normalized to that found in the non-transgenic littermates.Several of the one-year-old non-transgenic control mice were found to beobese, and at least one of the controls exhibited changes consistentwith the development of type II diabetes. However, none of theone-year-old transgenic mice were obese or showed any evidence ofdeveloping diabetes. Thus, it appears that FGF-like polypeptidetransgenics do not develop at least some of the age related changescommonly seen in mice as they age and, indeed, that the FGF-like gene ofthis invention may help retard the aging process.

These findings are significant and support the conclusion that theFGF-like polynucleotides and polypeptides of the present invention maybe useful for the treatment or diagnosis of age-related diseases,disorders, or conditions. By way of illustration, such diseases,disorders or conditions may include, without limitation,atherosclerosis, hypercholesterolemia, diabetes, obesity, stroke,osteoporosis, osteoarthritis, degenerative joint disease, muscleatrophy, sarcopenia, decreased lean body mass, baldness, wrinkles,increased fatigue, decreased stamina, decreased cardiac function, immunesystem dysfunction, cancer, Parkinson's disease, senile dementia,Alzheimer's disease, and decreased cognitive function. More generally,the molecules of the present invention may be applicable for enhancingor increasing life-span.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1. A method for treating obesity comprising administering to a humanpatient in need thereof: (a) an isolated polypeptide comprising: (i) anamino acid sequence that is that is at least 95% identical to the aminoacid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5; (ii) a fragment ofthe amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5 comprising atleast 150 amino acid residues; or (iii) an amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 5 having a C- and/or N-terminal truncation, whereinthe polypeptide comprises at least 150 amino acid residues; (b) anisolated polypeptide comprising an amino acid sequence that is encodedby: (i) a nucleotide sequence that is at least 95% identical to thenucleotide sequence of either SEQ ID NO: 1 or the DNA insert in ATCCDeposit No. PTA-626; (ii) a nucleotide sequence encoding a polypeptidethat is at least 95% identical to the polypeptide of either SEQ ID NO: 2or SEQ ID NO: 5; or (iii) a region of the nucleotide sequence of eitherSEQ ID NO: 1 or the DNA insert in ATCC Deposit No. PTA-626, encoding apolypeptide fragment of at least 150 amino acid residues; (c) apharmaceutical composition comprising an isolated polypeptidecomprising: (i) an amino acid sequence that is that is at least 95%identical to the amino acid sequence of either SEQ ID NO: 2 or SEQ IDNO: 5; (ii) a fragment of the amino acid sequence of SEQ ID NO: 2 or SEQID NO: 5 comprising at least 150 amino acid residues; or (iii) an aminoacid sequence of SEQ ID NO: 2 or SEQ ID NO: 5 having a C- and/orN-terminal truncation, wherein the polypeptide comprises at least 150amino acid residues; and a pharmaceutically acceptable formulationagent; or (d) a pharmaceutical composition comprising an isolatedpolypeptide comprising an amino acid sequence that is encoded by: (i) anucleotide sequence that is at least 95% identical to the nucleotidesequence of either SEQ ID NO: 1 or the DNA insert in ATCC Deposit No.PTA-626; (ii) a nucleotide sequence encoding a polypeptide that is atleast 95% identical to the polypeptide of either SEQ ID NO: 2 or SEQ IDNO: 5; or (iii) a region of the nucleotide sequence of either SEQ ID NO:1 or the DNA insert in ATCC Deposit No. PTA-626, encoding a polypeptidefragment of at least 150 amino acid residues; and a pharmaceuticallyacceptable formulation agent. 2-4. (canceled)
 5. A method for treatingobesity comprising administering to a human patient in need thereof: (a)a polypeptide produced by culturing a recombinant host cell undersuitable conditions to express the polypeptide and isolating thepolypeptide, wherein the recombinant host cell comprises a nucleic acidmolecule encoding a polypeptide comprising: (i) an amino acid sequencethat is that is at least 95% identical to the amino acid sequence ofeither SEQ ID NO: 2 or SEQ ID NO: 5; (ii) a fragment of the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 5 comprising at least 150 aminoacid residues; or (iii) an amino acid sequence of SEQ ID NO: 2 or SEQ IDNO: 5 having a C- and/or N-terminal truncation, wherein the polypeptidecomprises at least 150 amino acid residues; (b) a polypeptide producedby culturing a recombinant host cell under suitable conditions toexpress the polypeptide and isolating the polypeptide, wherein therecombinant host cell comprises a nucleic acid molecule comprising: (i)a nucleotide sequence that is at least 95% identical to the nucleotidesequence of either SEQ ID NO: 1 or the DNA insert in ATCC Deposit No.PTA-626; (ii) a nucleotide sequence encoding a polypeptide that is atleast 85% 95% identical to the polypeptide of either SEQ ID NO: 2 or SEQID NO: 5; or (iii) a region of the nucleotide sequence of either SEQ IDNO: 1 or the DNA insert in ATCC Deposit No. PTA-626, encoding apolypeptide fragment of at least 150 amino acid residues; (c) apharmaceutical composition comprising a polypeptide produced by aprocess in which a recombinant host cell is cultured under suitableconditions to express the polypeptide and isolating the polypeptide,wherein the recombinant host cell comprises a nucleic acid moleculeencoding a polypeptide comprising: (i) an amino acid sequence that isthat is at least 95% identical to the amino acid sequence of either SEQID NO: 2 or SEQ ID NO: 5; (ii) a fragment of the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 5 comprising at least 150 amino acidresidues; or (iii) an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:5 having a C- and/or N-terminal truncation, wherein the polypeptidecomprises at least 150 amino acid residues; and a pharmaceuticallyacceptable formulation agent; or (d) a pharmaceutical compositioncomprising a polypeptide produced by a process in which a recombinanthost cell is cultured under suitable conditions to express thepolypeptide and the polypeptide is isolated, wherein the recombinanthost cell comprises a nucleic acid molecule comprising: (i) a nucleotidesequence that is at least 95% identical to the nucleotide sequence ofeither SEQ ID NO: 1 or the DNA insert in ATCC Deposit No. PTA-626; (ii)a nucleotide sequence encoding a polypeptide that is at least 95%identical to the polypeptide of either SEQ ID NO: 2 or SEQ ID NO: 5; or(iii) a region of the nucleotide sequence of either SEQ ID NO: 1 or theDNA insert in ATCC Deposit No. PTA-626, encoding a polypeptide fragmentof at least 150 amino acid residues; and a pharmaceutically acceptableformulation agent. 6-8. (canceled)
 9. The method of claims 1 (c), 1 (d),5(c) or 5(d), wherein the pharmaceutically acceptable formulation agentis a carrier, adjuvant, solubilizer, stabilizer, or anti-oxidant. 10.The method of claim 1 or 5, wherein the isolated polypeptide iscovalently modified with a water-soluble polymer.
 11. The method ofclaim 10, wherein the water-soluble polymer is polyethylene glycol(PEG), dextran, monomethoxy-polyethylene glycol, cellulose,poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycolhomopolymers, polypropylene oxidelethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol.
 12. The method of claim11, wherein the water-soluble polymer is PEG.
 13. The method of claims1, 5, or wherein the polypeptide is fused to a heterologous amino acidsequence.
 14. The method of claim 13, wherein the heterologous aminoacid sequence is an IgG constant domain or fragment thereof.